Follicle-stimulating hormone regulates expression and activity of epidermal growth factor receptor in the murine ovarian follicle

Fertility depends on the precise coordination of multiple events within the ovarian follicle to ensure ovulation of a fertilizable egg. FSH promotes late follicular development, including expression of luteinizing hormone (LH) receptor by the granulosa cells. Expression of its receptor permits the subsequent LH surge to trigger the release of ligands that activate EGF receptors (EGFR) on the granulosa, thereby initiating the ovulatory events. Here we identify a previously unknown role for FSH in this signaling cascade. We show that follicles of Fshb^−/− mice, which cannot produce FSH, have a severely impaired ability to support two essential EGFR-regulated events: expansion of the cumulus granulosa cell layer that encloses the oocyte and meiotic maturation of the oocyte. These defects are not caused by an inability of Fshb^−/− oocytes to produce essential oocyte-secreted factors or of Fshb^−/− cumulus cells to respond. In contrast, although expression of both Egfr and EGFR increases during late folliculogenesis in Fshb^+/− females, these increases fail to occur in Fshb^−/− females. Remarkably, supplying a single dose of exogenous FSH activity to Fshb^−/− females is sufficient to increase Egfr and EGFR expression and to restore EGFR-dependent cumulus expansion and oocyte maturation. These studies show that FSH induces an increase in EGFR expression during late folliculogenesis and provide evidence that the FSH-dependent increase is necessary for EGFR physiological function. Our results demonstrate an unanticipated role for FSH in establishing the signaling axis that coordinates ovulatory events and may contribute to the diagnosis and treatment of some types of human infertility.Fertility in mammals depends on the coordinated execution of multiple events within the fully grown ovarian follicle at the time of ovulation (1, 2). The oocyte undergoes meiotic maturation, during which it progresses to metaphase II of meiosis and acquires the ability to begin embryonic development (3). Concomitantly, the layer of granulosa cells (GCs) immediately surrounding the oocyte, termed the “cumulus,” undergoes a process termed “expansion,” which is required for sperm to penetrate this layer and reach the oocyte (4–7). At the perimeter of the follicle, an inflammatory response associated with rupture of the follicular wall permits the cumulus–oocyte complex (COC) to escape from the follicle and enter the oviduct where fertilization will occur. These events are triggered by the preovulatory release of luteinizing hormone (LH), which acts on LH receptors (LHCGR) on the mural GCs that line the interior wall of the fully grown follicle (8).Recent studies have identified a key downstream effector of LH activity at ovulation. Binding of LH to LHCGR triggers the release of the EGF-related peptides amphiregulin (AREG, betacellulin (BTC), and epiregulin (EREG) (9–11). These bind to EGF receptors (EGFRs) located on both the mural and cumulus GCs (12–19) and activate MAPK3/1 as well as other signaling networks (20–28). Considerable evidence supports the view that the EGFR signaling mediates many or most ovulatory events. First, the release of the EGFR ligands follows the LH surge but precedes the LH-dependent responses (9–11). Second, EGF and the EGFR ligands can induce cumulus expansion and oocyte maturation in vitro, independently of LH (9, 10, 20, 29). Third, these events are impaired in mice bearing a hypomorphic Egfr allele that reduces EGFR activity by about one-half and in mice in which Egfr has been selectively inactivated in GCs through a targeted mutation (22, 23). Thus, the activation of EGFR signaling in GCs of mature follicles appears to be a major effector of the ovulatory response to LH.FSH binds to receptors located on GCs and induces the expression of numerous genes, including Lhcgr (8, 30). Lhcgr expression is impaired substantially in mice that lack either FSH, because of targeted mutation of the Fshb gene that encodes its β-subunit, or the FSH receptor and in humans bearing spontaneous mutations; these individuals fail to ovulate (31–34). Thus, the ovulatory response to LH depends strictly on the prior FSH-dependent expression of Lhcgr, and in this manner FSH indirectly controls the LHCGR-regulated release of the EGFR ligands. We report here that FSH also drives an increase in EGFR expression during late folliculogenesis and provide evidence that this increase is essential to enable the ovulatory response to EGF. By coordinating the expression of EGFR and the release of its ligands, FSH endows full-grown follicles with the capacity to activate EGFR signaling at ovulation.     

Osteopontin expression by CD103? dendritic cells drives intestinal inflammation

Intestinal CD103⁻ dendritic cells (DCs) are pathogenic for colitis. Unveiling molecular mechanisms that render these cells proinflammatory is important for the design of specific immunotherapies. In this report, we demonstrated that mesenteric lymph node CD103⁻ DCs express, among other proinflammatory cytokines, high levels of osteopontin (Opn) during experimental colitis. Opn expression by CD103⁻ DCs was crucial for their immune profile and pathogenicity, including induction of T helper (Th) 1 and Th17 cell responses. Adoptive transfer of Opn-deficient CD103⁻ DCs resulted in attenuated colitis in comparison to transfer of WT CD103⁻ DCs, whereas transgenic CD103⁻ DCs that overexpress Opn were highly pathogenic in vivo. Neutralization of secreted Opn expressed exclusively by CD103⁻ DCs restrained disease severity. Also, Opn deficiency resulted in milder disease, whereas systemic neutralization of secreted Opn was therapeutic. We determined a specific domain of the Opn protein responsible for its CD103⁻ DC-mediated proinflammatory effect. We demonstrated that disrupting the interaction of this Opn domain with integrin α9, overexpressed on colitic CD103⁻ DCs, suppressed the inflammatory potential of these cells in vitro and in vivo. These results add unique insight into the biology of CD103⁻ DCs and their function during inflammatory bowel disease.Inflammatory bowel diseases (IBDs), including Crohn disease (CD) and ulcerative colitis (UC), are caused by excessive inflammatory responses to commensal microflora and other antigens present in the intestinal lumen (1). Intestinal dendritic cells (DCs) contribute to these inflammatory responses during human IBD, as well as in murine colitis models (2). DCs that reside in draining mesenteric lymph nodes (MLNs) are also crucial mediators of colitis induction (3) and may be grouped based on their surface CD103 (integrin αE) expression as CD11c^highCD103⁺ (CD103⁺ DCs) and CD11c^highCD103⁻ (CD103⁻ DCs) (4–6). CD103⁺ DCs are considered important mediators of gut homeostasis in steady state (4, 5, 7–9), and their tolerogenic properties are conserved between mice and humans (5). However, their role during intestinal inflammation is not well defined. Instead, CD103⁻ DC function has been described mostly during chronic experimental colitis (10–12). These cells secrete IL-23, IL-6, and IL-12 (10–12), contributing to the development of T helper (Th) 17 and Th1 cells, and are highly inflammatory during CD4⁺ T-cell transfer colitis (12) and during 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis (11). MLN CD103⁻ DCs cultured in the presence of LPS, a Toll-like receptor (TLR) 4 agonist, or R848, a TLR7 agonist, express higher levels of TNF-α and IL-6 (7, 12). In fact, these cells secrete IL-23 and IL-12 even in the absence of TLR stimulation (10). Both MLN CD103⁻ and CD103⁺ DC subsets are present in acute colitis (11, 13); however, their function, as well as their cytokine profile, during this phase of disease, reflecting colitis initiation, remains unknown.Recent studies suggest a proinflammatory role for the cytokine osteopontin (Opn) in TNBS- and dextran sulfate sodium (DSS)-induced colitis (14, 15), which are the models for CD and UC, respectively. Opn is expressed by DCs and other immune cell types, such as lymphocytes, during autoimmune responses (16–22), and its expression by DCs during autoimmunity contributes to disease severity (17–19, 21, 23). In addition, Opn expression is highly up-regulated in intestinal immune and nonimmune cells and in the plasma of patients with CD and UC (24–29), as well as in the colon and plasma of mice with experimental colitis (14, 15, 27, 30). Increased plasma Opn levels are related to the severity of CD inflammation (29), and certain Opn gene (Spp1) haplotypes are modifiers of CD susceptibility (31), indicating that Opn could be used as an IBD biomarker (27). In general, Opn affects DC biology during several inflammatory conditions (17–21, 32–37) and could be a potential therapeutic target in IBD.In this study, we initially asked whether Opn was expressed by MLN CD103⁻ and CD103⁺ DCs during colitis. We found that CD103⁻ DCs express excessive levels of Opn in addition to other proinflammatory cytokines. Conversely, CD103⁺ DCs express profoundly lower levels of Opn and are noninflammatory. Using adoptive transfer of purified specific DC subsets, we determined that MLN CD103⁻ DCs are critical mediators of acute intestinal inflammation and that their Opn expression is essential for their proinflammatory properties in both acute and chronic colitis. Furthermore, Opn-deficient and Opn-neutralized mice developed significantly milder disease. In addition, we constructed transgenic (Tg) mice overexpressing Opn only in DCs. These mice developed exaggerated colitis, and adoptive transfer of their CD103⁻ DCs into recipient mice dramatically exacerbated disease. Because Opn protein contains several domains interacting with various receptors, we defined a specific Opn domain significant for inducing proinflammatory properties in CD103⁻ DCs. Blockade of the interaction of this Opn domain [containing functional Ser-Leu-Ala-Tyr-Gly-Leu-Arg (SLAYGLR) sequence] with integrin α9 expressed on CD103⁻ DCs abrogated their proinflammatory profile and colitogenic effects in vivo.     

Angptl4 links α-cell proliferation following glucagon receptor inhibition with adipose tissue triglyceride metabolism

Type 2 diabetes is characterized by a reduction in insulin function and an increase in glucagon activity that together result in hyperglycemia. Glucagon receptor antagonists have been developed as drugs for diabetes; however, they often increase glucagon plasma levels and induce the proliferation of glucagon-secreting α-cells. We find that the secreted protein Angiopoietin-like 4 (Angptl4) is up-regulated via Pparγ activation in white adipose tissue and plasma following an acute treatment with a glucagon receptor antagonist. Induction of adipose angptl4 and Angptl4 supplementation promote α-cell proliferation specifically. Finally, glucagon receptor antagonist improves glycemia in diet-induced obese angptl4 knockout mice without increasing glucagon levels or α-cell proliferation, underscoring the importance of this protein. Overall, we demonstrate that triglyceride metabolism in adipose tissue regulates α-cells in the endocrine pancreas.Type 2 diabetes is a metabolic disease characterized by high levels of fasting and postprandial glucose levels. Diabetic patients display elevated levels of glucagon and a relatively low activity of insulin, leading to increased hepatic gluconeogenesis, reduced glucose uptake, and altered lipid profile (1–4). At the histological level, islets of diabetic patients display an increase in the numbers of glucagon-secreting α-cells and a decrease in the number of insulin-secreting β-cells (5–7). Glucagon has received increasing attention after glucagon receptor knockout mice (gcgr^−/−) were shown to be protected from development of diabetes in type 1 and type 2 diabetes models (8, 9). These and other studies highlight diabetes as a joint glucagon and insulin disorder (10–12).Glucagon receptor antagonists (GRAs) have been developed as antidiabetic drugs. GRAs improve glycemic control in humans, but may induce compensatory hyperglucagonemia and proliferation of α-cells (13–15). These results concur with the dramatic hyperglucagonemia and increase in α-cell proliferation in gcgr^−/− mice (16) and humans with a nonfunctional glucagon receptor (17). The adverse side effects of GRAs present a practical need to understand the compensatory response of α-cells and raise basic questions regarding the control over α-cell proliferation. Surprisingly, nearly full ablation of α-cells does not increase α-cell proliferation or alter circulating glucagon levels (18), raising the hypothesis that, unlike β-cells, hormonal hypersecretion alone does not promote proliferation (19, 20). Rather, a reduction of glucagon signaling, either by GRA treatment or receptor knockout, feeds back to induce α-cell proliferation (21).In this study, we treated mice with a GRA to identify secreted factors leading to α-cell proliferation and hyperglucagonemia. We find that Angptl4 is up-regulated in white adipose tissue (WAT) and in plasma following GRA treatment. Angptl4 is a multifunctional secreted protein that is cleaved into an N-terminal part containing a coil-coil domain that inhibits lipoprotein lipase (LPL) and a C-terminal part with a fibrinogen-like domain that affects vasculature (22). The LPL inhibitory N-terminal fragment constitutes most of the blood-borne fraction of Angptl4 and can act in a paracrine and endocrine manner (23, 24). Angptl4 is a glucocorticoid and Ppar target gene, up-regulated during fasting and exercise and expressed in many tissues, but primarily in WAT in mice. Local up-regulation of Angptl4 expression diverts triglyceride utilization for fatty acid oxidation to other tissues (25–30). Knockout and overexpression of angptl4 lead to decreased or increased triglyceride levels, respectively, in mice (31), and mutations in the human angptl4 gene are associated with lower triglyceride levels in the blood (32).We show that treatment with recombinant Angptl4 protein specifically increases α-cell proliferation rates of young and old mice without increasing glucagon levels. Activation of Pparγ up-regulates angptl4 expression in WAT but not in the liver and results in increased α-cell proliferation. Pparα activation increased hepatic angptl4 but did not raise α-cell proliferation rates. Notably, GRA treatment led to Pparγ activation in WAT but did not activate Pparα in liver. Caloric restriction, which increases plasma Angptl4 levels (29), led to up-regulation of WAT but not liver angptl4 expression and increased α-cell proliferation. Angptl4^−/− mice have a normal islet morphology and α-cell proliferation rate. GRA treatment improves glycemia of diet-induced obese (DIO) angptl4^−/− mice without increasing glucagon levels or α-cell proliferation. In all, the data show that Angplt4 is sufficient to induce α-cell proliferation and is required for the adverse response of α-cells to GRA treatment.     

Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala

Stimulating presynaptic terminals can increase the proton concentration in synapses. Potential receptors for protons are acid-sensing ion channels (ASICs), Na⁺- and Ca²⁺-permeable channels that are activated by extracellular acidosis. Those observations suggest that protons might be a neurotransmitter. We found that presynaptic stimulation transiently reduced extracellular pH in the amygdala. The protons activated ASICs in lateral amygdala pyramidal neurons, generating excitatory postsynaptic currents. Moreover, both protons and ASICs were required for synaptic plasticity in lateral amygdala neurons. The results identify protons as a neurotransmitter, and they establish ASICs as the postsynaptic receptor. They also indicate that protons and ASICs are a neurotransmitter/receptor pair critical for amygdala-dependent learning and memory.Although homeostatic mechanisms generally maintain the brain’s extracellular pH within narrow limits, neural activity can induce transient and localized pH fluctuations. For example, acidification may occur when synaptic vesicles, which have a pH of ∼5.2–5.7 (1–3), release their contents into the synapse. Studies of mammalian cone photoreceptors showed that synaptic vesicle exocytosis rapidly reduced synaptic cleft pH by an estimated 0.2–0.6 units (4–6). Transient synaptic cleft acidification also occurred with GABAergic transmission (7). Some, but not all, studies also reported that high-frequency stimulation (HFS) transiently acidified hippocampal brain slices, likely as a result of the release of synaptic vesicle contents (8, 9). Neurotransmission also induces a slower, more prolonged alkalinization (10, 11). In addition to release of synaptic vesicle protons, neuronal and glial H⁺ and HCO₃⁻ transporters, channels, H⁺-ATPases, and metabolism might influence extracellular pH (10–12).ASICs are potential targets of reduced extracellular pH. ASICs are Na⁺-permeable and, to a lesser extent, Ca²⁺-permeable channels that are activated by extracellular acidosis (13–19). In the brain, ASICs consist of homotrimeric and heterotrimeric complexes of ASIC1a, ASIC2a, and ASIC2b. The ASIC1a subunit is required for acid-activation in the physiological range (>pH 5.0) (20, 21). Several observations indicate that ASIC are located postsynaptically. ASICs are located on dendritic spines. Although similar to glutamate receptors, they are also present on dendrites and cell bodies (20, 22–24). ASIC subunits interact with postsynaptic scaffolding proteins, including postsynaptic density protein 95 and protein interacting with C-kinase-1 (20, 24–29). In addition, ASICs are enriched in synaptosome-containing brain fractions (20, 24, 30).Although these observations raised the possibility that protons might be a neurotransmitter, postsynaptic ASIC currents have not been detected in cultured hippocampal neurons (31, 32), and whether localized pH transients might play a signaling role in neuronal communication remains unclear. In previous studies of hippocampal brain slices, extracellular field potential recordings suggested impaired hippocampal long-term potentiation (LTP) in ASIC1a^−/− mice (20), although another study did not detect an effect of ASIC1a (33). Another study using microisland cultures of hippocampal neurons suggested that the probability of neurotransmitter release increased in ASIC1a^−/− mice (32).Here, we tested the hypothesis that protons are a neurotransmitter and that ASICs are the receptor. Criteria to identify substances as neurotransmitters have been proposed (34). Beg and colleagues (35) used these criteria to conclude that protons are a transmitter released from Caenorhabditis elegans intestine to cause muscle contraction. Key questions about whether protons meet criteria for a neurotransmitter are: Does presynaptic stimulation increase the extracellular proton concentration? Do protons activate currents in postsynaptic cells? Can exogenously applied protons reproduce effects of endogenous protons? What is the postsynaptic proton receptor? We studied lateral amygdala brain slices because amygdala-dependent fear-related behavior depends on a pH reduction (36). In addition, ASICs are abundantly expressed there, and ASIC1a^−/− mice have impaired fear-like behavior (36–38).     

Hyperglycemia in rodent models of type 2 diabetes requires insulin-resistant alpha cells

To determine the role of glucagon action in diet-induced and genetic type 2 diabetes (T2D), we studied high-fat-diet–induced obese (DIO) and leptin receptor-defective (LepR^−/−) rodents with and without glucagon receptors (GcgRs). DIO and LepR^−/−,GcgR^+/+ mice both developed hyperinsulinemia, increased liver sterol response element binding protein 1c, and obesity. DIO GcgR^+/+ mice developed mild T2D, whereas LepR^−/−,GcgR^+/+ mice developed severe T2D. High-fat–fed (HFF) glucagon receptor-null mice did not develop hyperinsulinemia, increased liver sterol response element binding protein 1c mRNA, or obesity. Insulin treatment of HFF GcgR^−/− to simulate HFF-induced hyperinsulinemia caused obesity and mild T2D. LepR^−/−,GcgR^−/− did not develop hyperinsulinemia or hyperglycemia. Adenoviral delivery of GcgR to GcgR^−/−,LepR^−/− mice caused the severe hyperinsulinemia and hyperglycemia of LepR^−/− mice to appear. Spontaneous disappearance of the GcgR transgene abolished the hyperinsulinemia and hyperglycemia. In conclusion, T2D hyperglycemia requires unsuppressible hyperglucagonemia from insulin-resistant α cells and is prevented by glucagon suppression or blockade.The prevalence of type 2 diabetes (T2D) in the United States was 29.1 million in 2012, and 37% of adults were identified as prediabetic (1). T2D is now present on every continent (2). Despite the magnitude of this threat to world physical and fiscal health, our understanding of the pathogenic pathway is vague and is based largely on epidemiologic correlations. For example, the correlation between T2D and obesity is so high that most obese Americans can be considered prediabetic, but the precise mechanism of this relationship is unknown. Although the “lipotoxic” effects of ectopic lipids were first suggested in 1994 (3) to link diet-induced obesity to T2D and other components of the metabolic syndrome (3–11), the relationship between IR and T2D is still poorly understood. Proposed hypothetical links range from beta cell “glucotoxicity” (12) to the action of modifier genes (13) to failure of redox control (14).It has recently been shown that glucagon receptor-null mice remain normoglycemic and nonketotic despite total insulin deficiency but that transduction of a glucagon receptor cDNA into their liver makes them severely diabetic (15, 16). This proves that, whether or not insulin action is present, suppression of glucagon action prevents hyperglycemia. It has long been known that insulin suppression of glucagon regulates alpha cell secretion (17, 18). Although the presence of hyperglucagonemia was established unequivocally in type 1 diabetes (T1D) (15, 16), direct evidence that it is essential for the hyperglycemia of T2D is lacking. However, it has long been known that glucagon is elevated in T2D (17, 19, 20) and is resistant to suppression by insulin.     

Hematopoietic RIPK1 deficiency results in bone marrow failure caused by apoptosis and RIPK3-mediated necroptosis

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is recruited to the TNF receptor 1 to mediate proinflammatory signaling and to regulate TNF-induced cell death. RIPK1 deficiency results in postnatal lethality, but precisely why Ripk1^−/− mice die remains unclear. To identify the lineages and cell types that depend on RIPK1 for survival, we generated conditional Ripk1 mice. Tamoxifen administration to adult RosaCreER^T2Ripk1^fl/fl mice results in lethality caused by cell death in the intestinal and hematopoietic lineages. Similarly, Ripk1 deletion in cells of the hematopoietic lineage stimulates proinflammatory cytokine and chemokine production and hematopoietic cell death, resulting in bone marrow failure. The cell death reflected cell-intrinsic survival roles for RIPK1 in hematopoietic stem and progenitor cells, because Vav-iCre Ripk1^fl/fl fetal liver cells failed to reconstitute hematopoiesis in lethally irradiated recipients. We demonstrate that RIPK3 deficiency partially rescues hematopoiesis in Vav-iCre Ripk1^fl/fl mice, showing that RIPK1-deficient hematopoietic cells undergo RIPK3-mediated necroptosis. However, the Vav-iCre Ripk1^fl/fl Ripk3^−/− progenitors remain TNF sensitive in vitro and fail to repopulate irradiated mice. These genetic studies reveal that hematopoietic RIPK1 deficiency triggers both apoptotic and necroptotic death that is partially prevented by RIPK3 deficiency. Therefore, RIPK1 regulates hematopoiesis and prevents inflammation by suppressing RIPK3 activation.The proinflammatory cytokine TNF stimulates receptor-interacting serine/threonine-protein kinase 1 (RIPK1) ubiquitination, NFκB and MAPK activation, and induction of apoptosis or necroptosis (1, 2). TNF signaling via TNF receptor 1 (TNFR1) is highly regulated and results in the recruitment of several adapter proteins including TNFR1-associated death domain (TRADD) protein, the E3 ubiquitin ligases cellular inhibitor of apoptosis protein-1 and -2 (cIAP1/2), and TNFR-associated factor 2 (TRAF2) or 5, and the serine threonine death domain-containing kinase RIPK1 (complex I) (1). We have demonstrated that the kinase activity of RIPK1 is not required for NFκB activation (3); rather, RIPK1 is modified by the addition of Lys63-linked and linear polyubiquitin chains (3–6). Polyubiquitinated RIPK1 then recruits NEMO/IκB kinase-γ (IKKγ) to mediate IKK activation and TAK1/TAB2/3 to mediate MAPK activation, resulting in antiapoptotic and proinflammatory gene expression (7, 8). Deubiquitination of RIPK1 by cylindromatosis (CYLD) results in the formation of a cytosolic complex containing TRADD, Fas-associated death domain protein (FADD), caspase-8, and RIPK1 (complex IIa) (2). Caspase-8 cleaves and inactivates RIPK1 and CYLD and stimulates apoptosis (9–11). In the absence of caspase-8 or the presence of caspase inhibitors, TNF family members and potentially other ligands stimulate the kinase activity of RIPK1 to induce necroptosis (9, 11–16). RIPK1 also is recruited to the Toll-like receptor adapter TRIF via the Rip homotypic interaction motif (RHIM) to mediate NFκB activation (17) and, under conditions of caspase-8 inhibition, initiates necroptosis (14, 16). Necrostatin-1 (Nec-1), an allosteric RIPK1 inhibitor, inhibits necroptosis induced by TNF or the TLR3 ligand poly I:C and abolishes the formation and activation of an RIPK1/3 complex (13–16, 18). Although the molecular details whereby RIPK1 initiates necroptosis are unclear, RIPK3 and the pseudo kinase MLKL appear to be required (2).Genetic studies in mice have revealed cross-regulation between the apoptotic and necroptotic pathways. For example, the FADD/caspase-8/FLICE-like inhibitory protein long form (FLIP_L) complex regulates RIPK1 and RIPK3 activity during development, because the embryonic lethality associated with a caspase-8 deficiency is completely rescued by the absence of RIPK3 (19, 20). Similarly, RIPK1 deficiency rescues FADD-associated embryonic lethality (21). Thus, in the absence of FADD or caspase-8, embryos succumb to RIPK1- and RIPK3-dependent necroptosis. However, Fadd^−/−/Ripk1^−/− mice, die perinatally (21, 22), as do Ripk1^−/− mice, revealing that RIPK1 has prosurvival roles beyond the regulation of the FADD/caspase-8/FLIP_L complex.We have demonstrated that complete RIPK1 deficiency results in increased TNF-induced cell death that can be rescued, in part, by the absence of the TNFR1 (22, 23). However, Ripk1^−/−Tnfr1^−/− animals still succumb (23), indicating that other death ligands/pathways contribute to the RIPK1-associated lethality. Consistent with this hypothesis, RIPK3 deficiency recently has been shown to rescue the perinatal lethality observed in Ripk1^−/−Tnfr1^−/− mice (24, 25). Similarly, combined caspase-8 and RIPK3 deficiency also rescues the RIPK1-associated lethality (24–26). Collectively, these genetic studies in mice reveal that the perinatal death of Ripk1^−/− mice reflects TNF-induced apoptosis and RIPK3-mediated necroptosis. The nature of the ligand(s) or the trigger(s) of RIPK3-mediated necroptosis in vivo remain unclear. However, Ripk1^−/− MEFs are prone to necroptosis induced by poly I:C or by treatment with type I or type II IFN (24, 25), suggesting that these pathways contribute. Although these studies reveal a regulatory role for RIPK1, the multiorgan cell death and inflammation observed in the complete and compound RIPK1-knockout strains have made it difficult to discern the specific tissues that require RIPK1 for survival.     

iRhoms 1 and 2 are essential upstream regulators of ADAM17-dependent EGFR signaling

The metalloproteinase ADAM17 (a disintegrin and metalloprotease 17) controls EGF receptor (EGFR) signaling by liberating EGFR ligands from their membrane anchor. Consequently, a patient lacking ADAM17 has skin and intestinal barrier defects that are likely caused by lack of EGFR signaling, and Adam17^−/− mice die perinatally with open eyes, like Egfr^−/− mice. A hallmark feature of ADAM17-dependent EGFR ligand shedding is that it can be rapidly and posttranslationally activated in a manner that requires its transmembrane domain but not its cytoplasmic domain. This suggests that ADAM17 is regulated by other integral membrane proteins, although much remains to be learned about the underlying mechanism. Recently, inactive Rhomboid 2 (iRhom2), which has seven transmembrane domains, emerged as a molecule that controls the maturation and function of ADAM17 in myeloid cells. However, iRhom2^−/− mice appear normal, raising questions about how ADAM17 is regulated in other tissues. Here we report that iRhom1/2^−/− double knockout mice resemble Adam17^−/− and Egfr^−/− mice in that they die perinatally with open eyes, misshapen heart valves, and growth plate defects. Mechanistically, we show lack of mature ADAM17 and strongly reduced EGFR phosphorylation in iRhom1/2^−/− tissues. Finally, we demonstrate that iRhom1 is not essential for mouse development but regulates ADAM17 maturation in the brain, except in microglia, where ADAM17 is controlled by iRhom2. These results provide genetic, cell biological, and biochemical evidence that a principal function of iRhoms1/2 during mouse development is to regulate ADAM17-dependent EGFR signaling, suggesting that iRhoms1/2 could emerge as novel targets for treatment of ADAM17/EGFR-dependent pathologies.ADAM17 (a disintegrin and metalloprotease 17) is a membrane-anchored metalloproteinase that controls two major signaling pathways with important roles in development and disease, the EGF receptor (EGFR) pathway and the proinflammatory tumor necrosis factor α (TNF-α) pathway (1–5). Mice lacking ADAM17 resemble mice with defects in EGFR signaling in that they have open eyes at birth, enlarged semilunar heart valves, and enlarged hypertrophic zones in long bone growth plates, most likely caused by a lack of ADAM17-dependent release of the EGFR ligands transforming growth factor α (TGF-α) and heparin-binding epidermal growth factor (HB-EGF) (3, 6–14). In humans, defects in skin and intestinal barrier protection have been reported in a patient lacking ADAM17 (15) and in patients treated with EGFR inhibitors (16, 17), and similar skin defects were recently identified in a patient with defective EGFR signaling (18). Mouse models of ADAM17/EGFR signaling appear to recapitulate these mechanisms, because defects in skin barrier protection can be observed by inactivating either ADAM17 or the EGFR in keratinocytes (19), as well as in mice expressing very low levels of ADAM17, which also have increased susceptibility to intestinal inflammation (20). A hallmark feature of ADAM17 is its rapid response to various activators of cellular signaling pathways (21–23), which is presumably important to allow a rapid response to injury and to maintain the skin and intestinal barrier. The rapid activation of ADAM17 is controlled by its transmembrane domain whereas the cytoplasmic domain is dispensable in this context (22), suggesting that ADAM17 is regulated by one or more other membrane proteins, yet the underlying mechanism has remained enigmatic.Recent studies have shown that the maturation and function of ADAM17 in myeloid cells depend on inactive Rhomboid 2 (iRhom2), a catalytically inactive member of the Rhomboid family of seven membrane-spanning intramembrane serine proteinases (24–28). Myeloid cells lacking iRhom2 release very little TNF-α in response to activation of Toll-like receptor 4 by lipopolysaccharide (LPS) (24, 26, 28). Therefore, mice lacking iRhom2 are protected from the detrimental effects of TNF-α in mouse models for septic shock and inflammatory arthritis, similar to conditional knockout mice lacking ADAM17 in myeloid cells (11, 26, 29). However, iRhom2^−/− (iR2^−/−) mice are viable with no evident spontaneous pathological phenotypes (26, 29), whereas Adam17^−/− (A17^−/−) mice die shortly after birth (3). A major unresolved question has therefore been whether iRhom2 and the related iRhom1 are the long-sought-after regulators of the function of ADAM17-dependent EGFR signaling in vivo. Here we generate iRhom1^−/− (iR1^−/−) mice, which are viable and healthy, and report that iR1/2^−/− double knockout mice closely resemble mice lacking ADAM17 or the EGFR, providing the first genetic evidence, to our knowledge, that the principal function of iRhoms1/2 during mouse development is to control ADAM17/EGFR signaling.     

Two-photon microscopy reveals early rod photoreceptor cell damage in light-exposed mutant mice

Atrophic age-related and juvenile macular degeneration are especially devastating due to lack of an effective cure. Two retinal cell types, photoreceptor cells and the adjacent retinal pigmented epithelium (RPE), reportedly display the earliest pathological changes. Abca4^−/−Rdh8^−/− mice, which mimic many features of human retinal degeneration, allowed us to determine the sequence of light-induced events leading to retinal degeneration. Using two-photon microscopy with 3D reconstruction methodology, we observed an initial strong retinoid-derived fluorescence and expansion of Abca4^−/−Rdh8^−/− mouse rod cell outer segments accompanied by macrophage infiltration after brief exposure of the retina to bright light. Additionally, light-dependent fluorescent compounds produced in rod outer segments were not transferred to the RPE of mice genetically defective in RPE phagocytosis. Collectively, these findings suggest that for light-induced retinopathies in mice, rod photoreceptors are the primary site of toxic retinoid accumulation and degeneration, followed by secondary changes in the RPE.In recent years, dramatic progress has been made in discovering genetic and environmental factors contributing to retinal diseases. Imaging modalities such as scanning laser ophthalmoscopy (SLO) and optical coherence tomography along with classic histological methods and functional techniques, such as electroretinography (ERG) and electrophysiological recordings, have facilitated characterization of retinal defects (1–3). Concurrently, molecular understanding of the chemistry and biology of vision has paved the way for the first successful treatment of inherited retinal diseases, such as Leber congenital amaurosis (4–6) or the advanced exudative form of age-related macular degeneration (AMD) (7, 8). However, identifying the cell type where the pathology originates and understanding the underlying pathological mechanisms have remained a challenge, impeding progress toward development of therapies effective against several common retinal diseases.A tight interconnection between the neuronal retina and retinal pigmented epithelium (RPE) is essential for flow of nutrients, retinoids, and metabolic products (9, 10). Detachment of the retina from the RPE leads to rapid retinal atrophy in vivo. Because of this functional interrelationship between the RPE and photoreceptors and lack of well-developed experimental methodologies, it is difficult to assess which cells are initially affected by pathology in retinal diseases such as Stargardt disease or AMD. Even with suitable rodent models of blinding diseases, access to individual cell types in their native settings remains a challenge.To identify the sequence of degenerative processes in the retina initiated by brief intense light exposure, we first selected a mouse model that exhibits many features associated with human Stargardt disease and AMD—namely, Abca4^−/−Rdh8^−/− mice (11, 12). These genetically modified mice lack both the ATP-binding cassette transporter 4 (ABCA4) and the all-trans-retinol (ROL) dehydrogenase enzyme (RDH8). Both proteins are involved in the retinoid cycle, a metabolic sequence of chemical transformations needed to maintain continuous regeneration of the visual chromophore, 11−cis−retinal (11cRAL) from all-trans-retinal (atRAL), and both are also required for efficient clearance of atRAL upon its liberation from activated rhodopsin (13–17). Impaired clearance of atRAL is detrimental to retinal cells due to the high toxicity of this reactive aldehyde to all cell types (18). Clearance of atRAL is achieved by ABCA4, which transports atRAL from the disk lumen to the cytoplasmic space of photoreceptor outer segments (19) where RDHs, including RDH8, then reduce atRAL to ROL (11, 20). Defective ABCA4 function has been associated with both Stargardt disease (21) and AMD (22). Other than atRAL itself, condensation products of atRAL, including diretinoid-pyridinium-ethanolamine (A2E) formed in the RPE can also cause retinal degeneration (23). Formation of A2E is normally a relatively slow process requiring several biochemical reactions in photoreceptor and RPE cells (24, 25). A2E overaccumulation is observed in the RPE of individuals with Stargardt disease and is a risk factor for AMD. This by-product can thus serve as a marker of atRAL-associated changes and/or direct toxicity to the retina (1, 26–28). Identification of the primary cause of retinal degeneration, whether it is atRAL or its condensation products, as well as determination of which cell types are initially affected comprise two particularly intriguing questions that need answers to guide the development of optimal therapeutic interventions.Brief exposure of Abca4^−/−Rdh8^−/− mice to intense light results in acute retinal degeneration, which allows investigators to follow the precise sequence of degenerative events at both a cellular and molecular level (18, 29). Notably, such retinal degeneration can be prevented by inhibition of atRAL production with retinylamine, a retinoid cycle inhibitor (30), or by sequestration of atRAL by producing Schiff-base adducts of atRAL with drugs containing a primary amine group (12, 29). The Abca4^−/−Rdh8^−/− mouse also exhibits slowly progressive retinal degeneration under normal lighting conditions with a phenotype similar to AMD that responds to the above treatments (29). Downstream consequences of atRAL-induced cellular toxicity have also been studied, and pharmacologic inhibition of certain downstream targets can prevent atRAL-induced cell death (31).To monitor the flow of retinoids in the retinas of Abca4^−/−Rdh8^−/− mice after bright light exposure, we developed fluorescent imaging techniques with 3D reconstruction that take advantage of the fluorescent properties of certain isoprenoids and their condensation products. Noninvasive, high-resolution imaging of the retina was achieved by using two-photon microscopy (TPM), which offers real time, high-resolution images of endogenous fluorescent molecules in living tissues (32, 33). These methods, along with supplementary histological approaches, were used here to gain insights into the initiation of photoreceptor cell/RPE pathologies in Abca4^−/−Rdh8^−/− mice after bright light exposure.Now, we provide evidence indicating that light-induced production of atRAL in Abca4^−/−Rdh8^−/− mice causes RPE-independent degeneration of photoreceptor cells. Moreover, we show that active phagocytosis of affected photoreceptor cells by the RPE is required for the development of pathological changes in the RPE. Taken together, these results support a model whereby the primary site of pathology is photoreceptor cells, with RPE degeneration developing as a consequence of phagocytosis of excess atRAL condensation products accumulated primarily in rod outer segments (ROS) after light exposure.     

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2^−/− mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2′-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.Inflammatory bowel disease (IBD) is a chronic and relapsing intestinal inflammatory disease that arises through unknown genetic, environmental, and bacterial origins (1, 2). Ulcerative colitis (UC) and Crohn disease (CD) are the two main forms of IBD, and their incidence is increasing in industrialized countries (3). Furthermore, IBD is a risk factor for the development of colon cancer (4). Although the specific determinants remain elusive, persistent inflammation is believed to play a significant role in colon cancer development (5).Neutrophil recruitment and activation are key steps in the intestinal innate immune response observed in IBD (6–8), and studies with animal models of colitis highlight the relationship between neutrophil infiltration and disease severity (9–11). We recently reported results of a comprehensive analysis of histopathology, changes in gene expression, and nucleic acid damage occurring during progression of lower bowel disease in Rag2^−/− mice infected with Helicobacter hepaticus (Hh) (10). This mouse model emulates many aspects of human IBD, and infected mice develop severe colitis that progress into colon carcinoma, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages (12, 13).Phagocytes produce strong oxidants and radicals that damage cellular macromolecules and promote tissue damage at sites of inflammation (14–16). Myeloperoxidase (MPO) is an abundant enzyme in neutrophils that produces hypochlorous acid (HOCl) from hydrogen peroxide (H₂O₂) and chloride ion (17, 18). HOCl can oxidize and chlorinate DNA, proteins, and lipids (19, 20). A prominent target of HOCl is tyrosine, which leads to the formation of the stable aromatic residue, 3-chlorotyrosine (Cl-Tyr) (21, 22). MPO also produces chlorinating species that react with DNA to form chlorinated adducts such as 5-chloro-2′-deoxycytidine (5-Cl-dC) (23), the presence of which was identified in colon tissue of H. hepaticus-infected Rag2^−/− mice (10). This modification of DNA may provide a mechanistic link between neutrophil activity and colitis-associated carcinoma (10, 24, 25).Macrophages also contribute to the array of oxidants and radicals at sites of inflammation through release of nitric oxide (NO) generated by the inducible NO synthase (iNOS) enzyme. NO reacts with superoxide anion (O₂^−•) at diffusion-controlled rates to yield highly reactive peroxynitrite (ONOO⁻) (26, 27). MPO also reacts H₂O₂ with nitrite (NO₂⁻, the endpoint of cellular NO oxidation) to produce the strong nitrating agent, nitrogen dioxide radical (NO₂^•) (28). Both NO₂^• and ONOO⁻ can react with tyrosine residues to generate the stable tyrosine nitration product, 3-nitrotyrosine (Nitro-Tyr) (29, 30).Multiple MS methods have been applied for determination of Cl-Tyr and Nitro-Tyr levels in biological systems (10, 31–38), and both have been detected in inflamed tissues from animals and humans (11, 39). The presence of Nitro-Tyr has been demonstrated in colon tissue of IBD patients by immunohistochemistry, and levels were reported to correlate with disease activity (40, 41). We undertook the present study to test the null hypothesis that the H. hepaticus-infected mouse model of colitis and colitis-associated carcinoma represents a useful surrogate of human IBD. To examine this hypothesis, we first quantified levels of Nitro-Tyr and Cl-Tyr in proteins and 5-Cl-dC in DNA of colon tissues of IBD patients. Comparison of these data with our previous findings (10) further assessed the validity of this animal model. We then tested the hypothesis that inflammation-induced damage in the colon would be reflected in changes in serum constituents, and would therefore serve as a noninvasive measure of IBD activity. For this purpose, we determined levels of protein chlorination and nitration products, acute-phase proteins, cytokines, and chemokines in human and mouse sera. In addition, gene expression of several inflammatory signaling molecules was monitored in mice colons to determine whether colonic inflammation was directly associated with serum cytokine levels. We then used multivariate analysis to determine which systemic inflammatory markers in serum were most closely associated with disease activity and were also common to human IBD and H. hepaticus-associated colitis in Rag2^−/− mice.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Programmed cell death 1 inhibits inflammatory helper T-cell development through controlling the innate immune response

Programmed cell death 1 (PD-1) is an inhibitory coreceptor on immune cells and is essential for self-tolerance because mice genetically lacking PD-1 (PD-1^−/−) develop spontaneous autoimmune diseases. PD-1^−/− mice are also susceptible to severe experimental autoimmune encephalomyelitis (EAE), characterized by a massive production of effector/memory T cells against myelin autoantigen, the mechanism of which is not fully understood. We found that an increased primary response of PD-1^−/− mice to heat-killed mycobacteria (HKMTB), an adjuvant for EAE, contributed to the enhanced production of T-helper 17 (Th17) cells. Splenocytes from HKMTB-immunized, lymphocyte-deficient PD-1^−/− recombination activating gene (RAG)2^−/− mice were found to drive antigen-specific Th17 cell differentiation more efficiently than splenocytes from HKMTB-immunized PD-1^+/+ RAG2^−/− mice. This result suggested PD-1’s involvement in the regulation of innate immune responses. Mice reconstituted with PD-1^−/− RAG2^−/− bone marrow and PD-1^+/+ CD4⁺ T cells developed more severe EAE compared with the ones reconstituted with PD-1^+/+ RAG2^−/− bone marrow and PD-1^+/+ CD4⁺ T cells. We found that upon recognition of HKMTB, CD11b⁺ macrophages from PD-1^−/− mice produced very high levels of IL-6, which helped promote naive CD4⁺ T-cell differentiation into IL-17–producing cells. We propose a model in which PD-1 negatively regulates antimycobacterial responses by suppressing innate immune cells, which in turn prevents autoreactive T-cell priming and differentiation to inflammatory effector T cells.Autoimmune disease development is impacted by both genetic and environmental factors. Programmed cell death 1 (PD-1) is a type I membrane protein that delivers inhibitory signals to immune cells upon the binding of its ligand, PD-L1 or PD-L2 (1). PD-1 has been shown to be important for self-tolerance because spontaneous autoimmune diseases develop in PD-1^−/− mice (2–4). A single-nucleotide polymorphism that affects PD-1 expression is associated with autoimmune diseases in humans, such as systemic lupus erythematosus (5), type I diabetes (6), rheumatoid arthritis (7), and multiple sclerosis (MS) (8), suggesting that PD-1 deficiency may be a genetic factor involved in the development of autoimmunity.Experimental autoimmune encephalomyelitis (EAE) is a rodent model of T-cell–mediated inflammatory disease in the central nervous system (CNS), causing demyelination, axonal damage, and paralysis, and is a commonly used model for human MS. Previous reports suggested that PD-1 functions to attenuate EAE. PD-1 and its ligands were found to be strongly expressed on immune infiltrates in the CNS during the peak phase of EAE (9–11). In EAE studies, PD-1–deficient mice or the use of blocking antibodies that inhibit PD-1 engagement by ligands resulted in earlier disease onset, increased inflammatory infiltrates, and increased severity of clinical symptoms compared with normal disease progression (10–16). It has been demonstrated that ligand engagement of PD-1 inhibits T-cell activation, expansion, and cytokine production (17–19). Similarly, in EAE, PD-1 signaling in CNS-specific helper T cells may inhibit their expansion and secretion of inflammatory cytokines (10–12). Recently, T-helper 17 (Th17) cells were shown to be involved in EAE by producing IL-17 and GM-CSF (20, 21). Two reports showed that PD-1^−/− mice mount an augmented Th17 response to EAE induction (14, 16). However, the fundamental mechanisms by which PD-1 regulates antigen-specific Th17 cell differentiation, expansion, and effector function in EAE remain to be understood.To induce EAE, mice are immunized with myelin autoantigens in an emulsion of Mycobacterium tuberculosis (MTB)-derived adjuvants, causing a strong innate inflammatory response, leading to Th skewing (22). Curiously, recent studies showed that PD-1^−/− mice exhibited an altered response to infection with mycobacteria, characterized by uncontrolled bacterial burden; massive production of cytokines, termed “cytokine storm”; and early death (23–25). We wondered if this unique response of PD-1^−/− mice to mycobacteria contributed to their Th response in EAE.In this study, we took a combination of genetic and immunological approaches in which the innate response to MTB-derived adjuvant and antigen-specific T-cell polarization were separately analyzed. The present data suggest that an enhanced innate response of PD-1^−/− mice to MTB contributes to the susceptibility of these mice to severe EAE. We propose a previously undescribed function of PD-1 in controlling the basal state of the innate immune response, the failure of which can cause the activation of adaptive immune responses, provoking autoimmunity.     

PML plays both inimical and beneficial roles in HSV-1 replication

After entry into the nucleus, herpes simplex virus (HSV) DNA is coated with repressive proteins and becomes the site of assembly of nuclear domain 10 (ND10) bodies. These small (0.1–1 μM) nuclear structures contain both constant [e.g., promyelocytic leukemia protein (PML), Sp100, death-domain associated protein (Daxx), and so forth] and variable proteins, depending on the function of the cells or the stress to which they are exposed. The amounts of PML and the number of ND10 structures increase in cells exposed to IFN-β. On initiation of HSV-1 gene expression, ICP0, a viral E3 ligase, degrades both PML and Sp100. The earlier report that IFN-β is significantly more effective in blocking viral replication in murine PML^+/+ cells than in sibling PML^−/− cells, reproduced here with human cells, suggests that PML acts as an effector of antiviral effects of IFN-β. To define more precisely the function of PML in HSV-1 replication, we constructed a PML^−/− human cell line. We report that in PML^−/− cells, Sp100 degradation is delayed, possibly because colocalization and merger of ICP0 with nuclear bodies containing Sp100 and Daxx is ineffective, and that HSV-1 replicates equally well in parental HEp-2 and PML^−/− cells infected at 5 pfu wild-type virus per cell, but poorly in PML^−/− cells exposed to 0.1 pfu per cell. Finally, ICP0 accumulation is reduced in PML^−/− infected at low, but not high, multiplicities of infection. In essence, the very mechanism that serves to degrade an antiviral IFN-β effector is exploited by HSV-1 to establish an efficient replication domain in the nucleus.Several prominent events take place after the entry of herpes simplex virus (HSV) DNA into the nucleus of newly infected cells. Thus, viral DNA becomes coated by repressive proteins, the function of which is to block viral gene expression (1–6); nuclear domain 10 (ND10) bodies colocalize with the viral DNA (7, 8); α or immediate early viral genes are expressed; and one viral protein, ICP0, degrades promyelocytic leukemia protein (PML) and Sp100, two key constituents of ND10 bodies in conjunction with the UbcH5A ubiquitin-conjugating enzyme (9–11). What is left of the ND10 bodies is infiltrated by viral proteins and becomes the viral replication compartment (12–15).ND10 bodies range between 0.1 and 1 μM in diameter. The composition of ND10 bodies varies depending on the cellular function or in response to stress, such as that resulting from virus infection (16–19). Among the constant components of ND10 are PML, Sp100, and death-domain associated protein (Daxx). PML has been reported to be critical for the recruitment of components and for the organization of the ND10 bodies (18–23). The function of ND10 bodies may vary under different cellular conditions and may also depend on their composition.A key question that remains unanswered is the function of ND10 bodies in infection, and in particular, why HSV has evolved a strategy that specifically targets PML and Sp100 for degradation. Two clues that may ultimately shed light on the function of ND10 is that exposure of cells to IFN leads to an increase in the number of ND10 bodies and an increase in PML (16, 24–26). The second clue emerged from the observation reported earlier by this laboratory is that pretreatment of murine PML^+/+ cells with IFN-β led to a drastic reduction in virus yields. In contrast, exposure of PML^−/− cells to IFN-β led to a significantly smaller decrease in virus yields (27). The results suggest PML is an antiviral effector of IFN-β, but many questions regarding the function of PML remain unanswered (28).In this study, we constructed a PML^−/− cell line (1D2) derived from HEp-2 cells. The first part of this report centers on the structure of ND10 bodies bereft of PML and the interaction of these bodies with ICP0. In the second part, we report on the replication of HSV-1 in PML^−/− cells. Here we show that HSV-1 replication and the accumulation of ICP0 are significantly reduced in PML^−/− cells exposed to low ratios of virus per cell. HSV has evolved a strategy to take advantage of PML before its degradation.     

The bone-sparing effects of estrogen and WNT16 are independent of each other

Wingless-type MMTV integration site family (WNT)16 is a key regulator of bone mass with high expression in cortical bone, and Wnt16^−/− mice have reduced cortical bone mass. As Wnt16 expression is enhanced by estradiol treatment, we hypothesized that the bone-sparing effect of estrogen in females is WNT16-dependent. This hypothesis was tested in mechanistic studies using two genetically modified mouse models with either constantly high osteoblastic Wnt16 expression or no Wnt16 expression. We developed a mouse model with osteoblast-specific Wnt16 overexpression (Obl-Wnt16). These mice had several-fold elevated Wnt16 expression in both trabecular and cortical bone compared with wild type (WT) mice. Obl-Wnt16 mice displayed increased total body bone mineral density (BMD), surprisingly caused mainly by a substantial increase in trabecular bone mass, resulting in improved bone strength of vertebrae L₃. Ovariectomy (ovx) reduced the total body BMD and the trabecular bone mass to the same degree in Obl-Wnt16 mice and WT mice, suggesting that the bone-sparing effect of estrogen is WNT16-independent. However, these bone parameters were similar in ovx Obl-Wnt16 mice and sham operated WT mice. The role of WNT16 for the bone-sparing effect of estrogen was also evaluated in Wnt16^−/− mice. Treatment with estradiol increased the trabecular and cortical bone mass to a similar extent in both Wnt16^−/− and WT mice. In conclusion, the bone-sparing effects of estrogen and WNT16 are independent of each other. Furthermore, loss of endogenous WNT16 results specifically in cortical bone loss, whereas overexpression of WNT16 surprisingly increases mainly trabecular bone mass. WNT16-targeted therapies might be useful for treatment of postmenopausal trabecular bone loss.Both estrogen and wingless-type MMTV integration site family (WNT)16 are crucial regulators of bone mass in women (1–5). The bone-sparing effect of estrogen is primarily mediated via estrogen receptor-α (ERα) (6). Estrogen-deficiency leads to rapid bone loss and contributes significantly to the development of postmenopausal osteoporosis that can be prevented by estradiol treatment. However, this treatment is associated with side effects such as breast cancer and thromboembolism (7, 8).The WNTs are a family of secreted glycoproteins that consists of 19 members in mammals, and which mediates autocrine and paracrine effects by binding to frizzled (Fzd) receptors and LDL-related protein 5/6 (LRP5/6) coreceptors (9). During the last decade, several lines of clinical and preclinical evidence have indicated that WNT signaling is critical in bone development and in the regulation of adult bone homeostasis (10–20) and modulation of WNT signaling has emerged as a promising strategy for increasing bone mass (21–23). Crosstalk and synergy between ERα signaling and the WNT pathways have been described (24–26). In the brain, estrogen signaling activates WNT by down-regulating dickkopf-1 (Dkk1), a WNT antagonist, to prevent neurodegeneration (27). In the uterus, estrogen prompts the canonical WNT signaling pathway in the uterine epithelium to induce uterine epithelial cell growth (28), and in breast cancer, ERα activation enhances cell growth via WNT signaling (29).Human genetic studies followed by subsequent mechanistic studies have recently revealed that WNT16 is a key physiological regulator of cortical bone mass and nonvertebral fracture risk (4, 5, 30–33). We recently demonstrated that WNT16 is osteoblast-derived and inhibits osteoclastogenesis both directly by acting on osteoclast progenitors and indirectly by increasing expression of osteoprotegerin (Opg) in osteoblasts (3). WNT16 is highly expressed in cortical bone but, for unknown reasons, only moderately expressed in trabecular bone. Experiments using WNT16-deleted mouse models clearly show that absence of WNT16 results in reduced cortical but not trabecular bone mass (3, 4, 30, 34). However, the effects of pharmacologically increased WNT16 levels on the cortical and trabecular bone compartments are unexplored. Furthermore, the role of WNT16 for the bone-sparing effect of estrogen is unknown. Herein we first demonstrate that the expression of WNT16 is enhanced by estradiol treatment, and we therefore hypothesized that the bone-sparing effects of estrogen are WNT16-mediated. This hypothesis was tested in mechanistic studies using two different genetically modified mouse models in which estrogen does not have the capacity to regulate WNT16 levels. Contradicting our hypothesis, we demonstrate that the bone-sparing effects of estrogen and WNT16 are independent of each other. In addition, unexpectedly, we demonstrate that overexpression of WNT16 increases mainly trabecular bone mass.     

Functions of vasopressin and oxytocin in bone mass regulation

Prior studies show that oxytocin (Oxt) and vasopressin (Avp) have opposing actions on the skeleton exerted through high-affinity G protein-coupled receptors. We explored whether Avp and Oxtr can share their receptors in the regulation of bone formation by osteoblasts. We show that the Avp receptor 1α (Avpr1α) and the Oxt receptor (Oxtr) have opposing effects on bone mass: Oxtr^−/− mice have osteopenia, and Avpr1α^−/− mice display a high bone mass phenotype. More notably, this high bone mass phenotype is reversed by the deletion of Oxtr in Oxtr^−/−:Avpr1α^−/− double-mutant mice. However, although Oxtr is not indispensable for Avp action in inhibiting osteoblastogenesis and gene expression, Avp-stimulated gene expression is inhibited when the Oxtr is deleted in Avpr1α^−/− cells. In contrast, Oxt does not interact with Avprs in vivo in a model of lactation-induced bone loss in which Oxt levels are high. Immunofluorescence microscopy of isolated nucleoplasts and Western blotting and MALDI-TOF of nuclear extracts show that Avp triggers Avpr1α localization to the nucleus. Finally, a specific Avpr2 inhibitor, tolvaptan, does not affect bone formation or bone mass, suggesting that Avpr2, which primarily functions in the kidney, does not have a significant role in bone remodeling.Over the past decade, we have described direct actions of anterior and posterior pituitary hormones on the skeleton (1–8). We have shown that these actions are exerted via G protein-coupled receptors resident on both osteoblasts and osteoclasts. We also find that the skeleton is highly sensitive to the action of posterior pituitary hormones; for example, mice haploinsufficient in oxytocin (Oxt) have osteopenic bones, but lactation is normal; lactation is impaired only in Oxt^−/− mice (2). Likewise, Tshr haploinsufficient mice are completely euthyroid with normal thyroid follicles but display significant osteopenia (4). The exquisite sensitivity of the skeleton to pituitary hormones comes as no surprise, considering that the pituitary gland and the skeleton are both evolutionarily more primitive than target endocrine organs (7).Apart from the known actions of growth hormone on the skeleton, Tsh, Fsh, Acth, Oxt, and vasopressin (Avp) have all been shown to regulate the formation and/or function of both osteoblasts and osteoclasts and thus to control bone remodeling in vivo (2–4, 6–8). The two neurohypophyseal hormones Oxt and Avp have opposing functions (2, 3). Oxt stimulates and Avp inhibits osteoblast formation. Consequently, the genetic deletion of the Oxt receptor (Oxtr) and Avp receptor 1α (Avpr1α) yields opposing phenotypes, notably osteopenia in Oxtr^−/− mice and high bone mass in Avpr1α^−/− mice (2, 3). These findings may explain the rapid recovery of bone loss at weaning when plasma Oxt levels are high (9) and also the profound loss of bone noted in chronic hyponatremic states, such as the syndrome of inappropriate antidiuretic hormone secretion (SIADH), in which serum Avp levels are elevated (3).We find high levels of Oxtr expression on both osteoclasts and osteoblasts (2, 10), in addition to their abundant expression in breast and uterine tissue, where they regulate lactation and parturition, respectively (11). Avpr1αs, in contrast, are distributed more ubiquitously, whereas Avpr2s are localized mainly in the kidney, where they regulate free water excretion (12). Osteoblasts express both Avpr1α and Avrpr2 (3). The only other known isoform, Avpr1β, is expressed predominantly in the pancreas and pituitary; it regulates ACTH secretion from pituitary corticotrophs (13). Sequence alignment shows that the binding sites of the Oxtr and Avprs are highly conserved, with specific amino acids within the predicted binding pocket providing ligand selectivity (14–16). The respective ligands Oxt and Avp also are homologous nonapeptides, differing in only two amino acids, and are known to interact with the other’s receptor with different affinities (17).To our knowledge, osteoblasts and osteoclasts are the only cells in which Oxtr, Avpr1α, and Avpr2 are coexpressed. We also have shown that osteoblastic Oxtrs undergo internalization and nuclear translocation upon binding to Oxt and that this action is independent of cytosolic Erk phosphorylation (18). Avpr1α activation by Avp also activates Erk phosphorylation within minutes (3). The homology between the ligands and their respective receptors and converging downstream signals suggest that Avp and Oxtr may share receptors with opposing or convergent signals. Here, we have explored these interactions in the regulation of osteoblastic bone formation by using mice lacking one or both receptors, chemical inhibitors, and physiological models of high bone turnover.     

Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability

Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ^−/− mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ^−/− mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ^−/− mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper–tyrosine-phosphorylated in the PTPσ^−/− mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ^−/− mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD.Protein tyrosine phosphatase (PTP)σ, encoded by PTPRS (1), consists of a cell adhesion molecule-like ectodomain containing three immunoglobulin (Ig)-like and three to eight fibronectin type III repeats, a transmembrane domain, and a cytosolic region with two PTPase domains, of which the first (D1) is catalytically active (2). PTPσ expression is developmentally regulated and found primarily in the nervous system and specific epithelia (3, 4). It was previously shown to play a role in axon growth and path finding (5–7), neuroregeneration (5, 8, 9), autophagy (10), and neuroendocrine development (11–13).To investigate the function of PTPσ in vivo, our group (11) and Tremblay and coworkers (12) generated PTPσ^−/− mice. These mice exhibited high neonatal mortality, various neurological and neuroendocrine defects, colitis, and cachexia (5, 11, 13, 14). Analysis of the intestinal tissue in surviving mice by our group revealed the presence of mucosal inflammation, intestinal crypt branching, and villus blunting: all features of colitis similar to the enteropathy associated with human inflammatory bowel disease (IBD) (15). Notably, PTPσ^−/− mice also showed increased susceptibility to chemical and infectious models of murine colitis, specifically treatment with dextran sodium sulfate (DSS) or infection with Citrobacter rodentium (15). The intestinal phenotype in the mice strongly inferred a connection between PTPσ and IBD.IBD is a chronic, idiopathic, relapsing disorder affecting the gastrointestinal tract, where Crohn disease and ulcerative colitis (UC) are the two major forms (16). In IBD pathogenesis, the presence of environmental factors together with polymorphisms in IBD-susceptibility genes cause an abnormal innate and adaptive host immune response to commensal gut bacteria, leading to sustained and deleterious inflammation (17). Chronic infection (18, 19), dysbiosis (19), defective mucosal barrier defense (20), and insufficient microbial clearance (19) have all been implicated as factors contributing to IBD pathogenesis. The disease is known to have a strong genetic component, as evidenced by specific populations exhibiting a disproportionately high incidence (21) and the high disease concordance between monozygotic twins (22). Genome-wide association studies and associated metaanalyses have implicated several genes and pathways in IBD, notably genes associated with intestinal barrier defense [MYO9B (23), PARD3 (24), MAGI2 (24), CDH1 (25, 26)].Through SNP analysis of IBD patients, we showed that PTPRS is genetically associated with UC (15). The identified SNP polymorphism leads to alternative splicing in the extracellular region of the epithelial isoform of PTPσ, causing loss of the third Ig domain (15). This splicing might potentially lead to altered ligand recognition or may affect receptor dimerization (27). In addition, through an interaction-trap assay, we identified the apical junction complex (AJC) proteins E-cadherin (CDH1) and β-catenin (CTNNB1) as colonic substrates of PTPσ (15). Interestingly, recent large-scale genetic studies have identified over 160 loci that affect risk of developing IBD, many of which involved in barrier regulation (24, 28, 29).The AJC confers polarity to epithelial cells and maintains intestinal barrier integrity (30). Defective regulation of AJC proteins creates disrupted epithelial barriers, permeability defects, and aberrant intestinal morphology (30, 31), similar to defects seen in IBD. The connection between the AJC and IBD is further demonstrated by our earlier SNP analysis, which revealed a haplotype polymorphism in CDH1 that is associated with Crohn disease, leading to a truncated E-cadherin protein that fails to localize to the plasma membrane (PM), as also observed in IBD patient biopsy samples (25). Thus, we postulate that PTPσ regulates epithelial barrier integrity through regulation of AJC proteins and that defective PTPσ function may contribute to IBD.In this report, we demonstrate an intestinal epithelial barrier defect in the PTPσ^−/− mice and identify the AJC protein ezrin as an in vivo colonic substrate for PTPσ. We further demonstrate that dephosphorylation of ezrin-Y353 or -Y145 by PTPσ leads to its redistribution from the PM to the cytosol, similar to its localization following induction of IBD in mice.     

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO₃⁻); it thereby serves as a cellular sensor for HCO₃⁻, carbon dioxide (CO₂), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO₃⁻ binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO₃⁻ activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO₃⁻ sensing and a basis for targeting this system with drugs.The ubiquitous second messenger cAMP regulates diverse physiological processes, from fungal virulence to mammalian brain function (1, 2). In mammals, cAMP can be generated by any of 10 differently expressed and regulated adenylyl cyclases (ACs): nine transmembrane enzymes (tmACs) and one soluble AC (sAC) (3). TmACs reside in the cell membrane, where they mediate cellular responses to hormones acting through G protein-coupled receptors (4). In contrast, sAC functions in various intracellular locations, providing cell-specific spatial and temporal patterns of cAMP (5–7) in response to intracellular signals, including calcium, ATP, and bicarbonate (HCO₃⁻) (3, 8–10). HCO₃⁻ regulation of sAC enzymes is a direct effect on their catalytic domains and is conserved across bacterial, fungal, and animal kingdoms (1, 11–13). Via modulation of sAC, and sAC-like cyclase activities, HCO₃⁻ serves as an evolutionarily conserved signaling molecule mediating cellular responses to HCO₃⁻, CO₂, and pH (3, 14). In mammals, sAC acts as a CO₂/HCO₃⁻/pH sensor in processes such as sperm activation (15), acid-base homeostasis (16), and various metabolic responses (10, 17, 18). sAC has also been implicated in skin and prostate cancer and as a target for male contraceptives (19–21).All mammalian ACs are class III nucleotidyl cyclases sharing homologous catalytic domains. Their catalytic cores are formed through symmetrical or pseudosymmetrical association of two identical or highly similar catalytic domains, C₁ and C₂ (22–24); in mammalian ACs, both domains reside on a single polypeptide chain. Such C₁C₂ pseudoheterodimers form two pseudosymmetrical sites at the dimer interface: the active site and a degenerated, inactive pocket (3, 23). A conserved Lys and an Asp/Thr in the active site recognize the base of the substrate ATP, and two conserved Asp residues bind two divalent cations, normally Mg²⁺ (23). The ions, called ion A and ion B, coordinate the substrate phosphates and support the intramolecular 3′-hydroxyl (3′-OH) attack at the α-phosphorous to form cAMP and pyrophosphate (PP_i) (3). In tmACs, the degenerate site binds forskolin (24), a plant diterpene that activates tmACs but has no effect on sAC (25). The forskolin activation mechanism and the existence of physiological ligands for this site in tmACs or in sAC remain unclear.There are two sAC isoforms known to be generated by alternative splicing (26). Full-length sAC comprises N-terminal catalytic domains along with ∼1,100 residues with a little understood function except for an autoinhibitory motif and a heme-binding domain (3, 27, 28). Exclusion of exon 12 (26) generates a truncated isoform, sAC_t (residues 1–490), which comprises just the two sAC catalytic domains (sAC-cat) (25). sAC_t is widely expressed, and it is the isoform most extensively biochemically characterized (3, 8, 11). It is directly activated by Ca²⁺ and HCO₃⁻; Ca²⁺ supports substrate binding, and HCO₃⁻ increases turnover and relieves substrate inhibition (8), and this regulation is conserved in sAC-like enzymes from Cyanobacteria to humans (3, 13, 29). In a homodimeric, HCO₃⁻-regulated sAC homolog from Spirulina platensis, adenylyl cyclase C (CyaC), HCO₃⁻ appeared to facilitate an active site closure required for catalysis (13), but the HCO₃⁻ binding site and its mechanism of activation remained unknown.Here, we present crystal structures of the human sAC-cat in apo form and in complex with substrate, products, bicarbonate, and a pharmacological inhibitor. The structures reveal insights into binding sites and mechanisms for sAC catalysis and for its regulation by physiological and pharmacological small molecules.