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.     

Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition

Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR undergoes conformational changes to dimerize, resulting in kinase activation and autophosphorylation and downstream signaling. Tyrosine kinase inhibitors (TKIs) have been used to treat lung cancer by inhibiting EGFR phosphorylation. Previously, we demonstrated that EGFR sialylation suppresses its dimerization and phosphorylation. In this report, we further investigated the effect of sialylation on the phosphorylation profile of EGFR in TKI-sensitive and TKI-resistant cells. Sialylation was induced in cancer progression to inhibit the association of EGFR with EGF and the subsequent autophosphorylation. In the absence of EGF the TKI-resistant EGFR mutant (L858R/T790M) had a higher degree of sialylation and phosphorylation at Y1068, Y1086, and Y1173 than the TKI-sensitive EGFR. In addition, although sialylation in the TKI-resistant mutants suppresses EGFR tyrosine phosphorylation, with the most significant effect on the Y1173 site, the sialylation effect is not strong enough to stop cancer progression by inhibiting the phosphorylation of these three sites. These findings were supported further by the observation that the L858R/T790M EGFR mutant, when treated with sialidase or sialyltransferase inhibitor, showed an increase in tyrosine phosphorylation, and the sensitivity of the corresponding resistant lung cancer cells to gefitinib was reduced by desialylation and was enhanced by sialylation.Epidermal growth factor receptor (EGFR), one of the most studied receptor tyrosine kinases, is a drug target for cancer therapy, because its kinase activity correlates with tumorigenicity (1). Under normal conditions, EGFR forms dimers upon ligand binding and induces kinase activation (2–6). The conformational change of EGFR from tethered to extended form induced by ligand binding involves the exposure of the interface, followed by dimerization, activation, and autophosphorylation (7). The phosphorylation code of EGFR determines the propensity of the downstream signaling network to regulate cell proliferation, survival, migration, and angiogenesis (8, 9).In a significant fraction of patients with nonsmall cell lung cancer (NSCLC), especially patients in Asia and those with the adenocarcinoma subtype, mutations in the kinase domain of EGFR cause constitutive activation and have been identified as an important factor in EGFR dysregulation (10, 11). Particularly, mutation from leucine to arginine at position 858 (L858R) and, less significantly, deletion of exon 19 that eliminates four amino acids (LREA) account for ∼90% of the mutations involved in the constitutive activation of EGFR. These mutations are commonly found in patients with increased sensitivity to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib (12–14). However, most patients with such mutations show resistance within months after TKI therapy, and >50% of them develop a second EGFR mutation, T790M, which confers TKI resistance by increasing the affinity for ATP and decreasing the affinity for TKIs (15–17).Studies have demonstrated that the glycans on EGFR participate in the regulation of EGFR function. The number of N-glycans and the degree of branching can regulate the cell-surface expression of EGFR in response to N-acetyl-d-glucosamine (GlcNAc) supplementation (18). In addition, studies with site-directed mutagenesis indicate that the glycans on Asn420 and 579 prevent EGFR from ligand-independent dimerization (19–21), and knocking down/out fucosyltransferase 8, the enzyme responsible for the core fucosylation, attenuates EGFR phosphorylation and EGF binding (22, 23). Moreover, our previous study revealed that sialylation and fucosylation suppress EGFR dimerization, autophosphorylation, and EGF-induced lung cancer cell invasion (24).Here, we investigated the effect of sialylation on EGFR dimerization to understand how extracellular sialylation influences intracellular phosphorylation in both wild-type and mutant EGFR. Our biochemical data demonstrated that sialylation could suppress EGFR dimerization by attenuating its association with EGF, and sialylation could significantly and selectively suppress tyrosine phosphorylation and affect the levels of phosphoserine and phosphothreonine on EGFR. In EGFR mutants, especially L858R/T790M, sialylation was observed to have a selective effect on EGFR phosphorylation, and inhibition of sialylation resulted in increased phosphorylation and resistance to gefitinib in this TKI-resistant lung cancer cell line. Further study of these findings should provide a better understanding of EGFR-mediated phosphorylation and disease progression affected by glycosylation and lead to the development of a new therapeutic strategy.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Inhibition of pancreatic carcinoma by homo- and heterocombinations of antibodies against EGF-receptor and its kin HER2/ErbB-2

Due to intrinsic aggressiveness and lack of effective therapies, prognosis of pancreatic cancer remains dismal. Because the only molecular targeted drug approved for pancreatic ductal adenocarcinoma is a kinase inhibitor specific to the epidermal growth factor receptor (EGFR), and this receptor collaborates with another kinase, called HER2 (human EGF-receptor 2), we assumed that agents targeting EGFR and/or HER2 would effectively retard pancreatic ductal adenocarcinoma. Accordingly, two immunological strategies were tested in animal models: (i) two antibodies able to engage distinct epitopes of either EGFR or HER2 were separately combined, and (ii) pairs of one antibody to EGFR and another to HER2. Unlike the respective single monoclonal antibodies, which induced weak effects, both types of antibody combinations synergized in animals in terms of tumor inhibition. Immunological cooperation may not depend on receptor density, antigenic sites, or the presence of a mutant RAS protein. Nevertheless, both types of antibody combinations enhanced receptor degradation. Future efforts will examine the feasibility of each strategy and the potential of combining them to achieve sustained tumor inhibition.Pancreatic cancer is the fourth leading cause of cancer death in western countries, with a 5-y survival of less than 10% (1). Genomic characterization of pancreatic ductal adenocarcinoma (PDAC), which accounts for over 90% of pancreatic cancer, identified multiple significantly mutated genes, including KRAS, TP53, CDKN2A, and SMAD4, and uncovered novel mutated genes (2). Advances in neoadjuvant and adjuvant chemotherapeutic regimens have resulted in some improvement in PDAC treatment outcome, but pancreatectomy remains the single most effective treatment modality for pancreatic cancer. A distinguishing molecular feature of PDAC is the presence of activating KRAS mutations in over 90% of tumors (3). Along with an ability to overcome inflammation-induced senescence (4), mutants of the RAS gene inevitably up-regulate a plethora of growth factors (e.g., TGF-alpha) and cytokines (e.g., interleukin-8), which likely contribute to disease progression. In line with this possibility, genetically engineered mouse models indicate that development of PDACs driven by KRAS is dependent on EGFR signaling (5). In the same vein, a small-molecule inhibitor of the epidermal growth factor receptor (EGFR) has been approved for the treatment of PDAC (6). Several studies reported high expression of EGFR, ranging from 7.7% to 100% of PDACs, but the abundance of ErbB-2/HER2, the oncogenic kin of EGFR, is relatively low (7). Notably, in response to ligand binding, EGFR forms heterodimers with HER2, and these complexes are characterized by enhanced signaling due to evasion of receptor endocytosis and degradation (8). Hence, simultaneous targeting of both EGFR and HER2 is a logical extension of the biochemistry of ErbB/HER signaling.Along with low molecular weight kinase inhibitors specific to EGFR and HER2, monoclonal antibodies (mAbs) against these receptors are routinely used in oncology wards to treat breast, gastric, colorectal, and head and neck carcinomas (9). Therapeutic antibodies may recruit the effector arm of the host cellular immune defense mechanism (10, 11). In addition, they might inhibit tumor cell proliferation by interfering with ligand binding, or by blocking receptor dimerization (12–14). An important feature of therapeutic anti-EGFR and anti-HER2 mAbs is their ability to collaborate with chemotherapeutic drugs (15, 16). However, another way to improve the efficacy of mAbs to surface receptors comprises combinations of two or more mAbs, each targeting a distinct receptor’s epitope. For example, it has been reported that certain pairs of anti-EGFR antibodies can accelerate receptor endocytosis and degradation (17), probably through a mechanism involving inhibition of receptor recycling (18). Consistent with these observations, a mixture of two anti-EGFR mAbs, called Sym004, inhibited cancer cell growth (14). Similarly, synergistic antitumor effects of mAbs directed to the rodent form of HER2 associated the therapeutic effect with enhanced receptor degradation (19), and synergistic effects mediated by the human HER2 protein were later confirmed (11, 20). However, both immune mechanisms involving recruitment of killer T cells (11) and nonimmune modes of action, involving growth arrest and receptor degradation (21), have been implicated in the mechanism underlying the antitumor effect of mAbs specific to HER2. Importantly, a mixture of two mAbs to HER2, trastuzumab and pertuzumab, in combination with chemotherapy, was found to significantly prolong progression-free survival of HER2-overexpressing breast cancer patients (22).The present study has been motivated by the lack of effective molecular targeted drugs to treat PDAC. We applied on xenografts of PDAC two immunological strategies: the first combined two antibodies to the same receptor, either EGFR or HER2, in similarity to our recent study that applied pairs of anti-EGFR antibodies on triple negative breast cancer (23). The other strategy combined two antibodies, one to EGFR and the other to HER2, in similarity to reports by Azria, Pelegrin, and coworkers, who combined two antibodies (24) and also added a third agent, namely a tyrosine kinase inhibitor (25). Here, we compare the two types of antibody combinations and also highlight potential mechanisms of the synergy observed in animals bearing human PDAC xenografts.     

Bisphosphonates inactivate human EGFRs to exert antitumor actions

Bisphosphonates are the most commonly prescribed medicines for osteoporosis and skeletal metastases. The drugs have also been shown to reduce cancer progression, but only in certain patient subgroups, suggesting that there is a molecular entity that mediates bisphosphonate action on tumor cells. Using connectivity mapping, we identified human epidermal growth factor receptors (human EGFR or HER) as a potential new molecular entity for bisphosphonate action. Protein thermal shift and cell-free kinase assays, together with computational modeling, demonstrated that N-containing bisphosphonates directly bind to the kinase domain of HER1/2 to cause a global reduction in downstream signaling. By doing so, the drugs kill lung, breast, and colon cancer cells that are driven by activating mutations or overexpression of HER1. Knocking down HER isoforms thus abrogates cell killing by bisphosphonates, establishing complete HER dependence and ruling out a significant role for other receptor tyrosine kinases or the enzyme farnesyl pyrophosphate synthase. Consistent with this finding, colon cancer cells expressing low levels of HER do not respond to bisphosphonates. The results suggest that bisphosphonates can potentially be repurposed for the prevention and therapy of HER family-driven cancers.Bisphosphonates are the mainstay of therapy worldwide for osteoporosis and skeletal metastasis (1, 2). However, the drugs have also been shown to kill cancer cells in people independently of their action on osteoclasts (3). Most compelling is evidence that breast cancer patients treated with the potent bisphosphonate zoledronic acid display a profound reduction in disseminated tumor cell burden and increased disease-free survival (4–7). More intriguing, however, is that patients on oral bisphosphonates for osteoporosis have a lower incidence of colon and breast cancer (8–10). Nonetheless, the mechanism underscoring these anticancer actions is not well understood.Whereas newer N-containing bisphosphonates inhibit farnesyl pyrophosphate synthase (FPPS) (1), they have also been shown to block tumor growth independently of FPPS, namely through ɤδ T-cell receptor activation (11–13), NF-κB inhibition (14), and VEGF and hypoxia inducible factor-1α suppression (15–17). However, the rank orders of the antitumor and anti-FPPS potencies of bisphosphonates do not match, suggesting that yet undiscovered mechanisms mediate their action on cancer cells. Here we report the human EGFR (HER) family of receptor tyrosine kinases (RTKs) as a potential molecular entity for bisphosphonate action.A large number of malignancies, including lung, breast, colorectal, stomach, head and neck, and pancreatic cancers, are driven by members of the human EGFR family, which are either overexpressed or mutated to constitutively active forms. About 30% of nonsmall-cell lung cancers, which cause the highest number of cancer-related deaths worldwide, are driven by activating mutations in the HER1 kinase domain, namely HER1^ΔE746-A750 and HER1^L868R (18). Although the tyrosine kinase inhibitors erlotinib and gefitinib improve survival of these patients (19), prolonged therapy invariably results in resistance (20). In contrast, HER2 (ErbB2/neu) is amplified in ∼25% of breast cancers and represents an aggressive subtype, constituting the second most common cause of cancer-related mortality (21). Additionally, up to 90% of colon cancers arise from HER1 gene amplification or protein overexpression (22). Here, we report that bisphosphonates bind to the kinase domain of HER1/2, inhibit global downstream signaling, and kill HER-driven lung, breast, and colon cancer cells.     

From the Cover: Phosphorylation of PCNA by EGFR inhibits mismatch repair and promotes misincorporation during DNA synthesis

Proliferating cell nuclear antigen (PCNA) plays essential roles in eukaryotic cells during DNA replication, DNA mismatch repair (MMR), and other events at the replication fork. Earlier studies show that PCNA is regulated by posttranslational modifications, including phosphorylation of tyrosine 211 (Y211) by the epidermal growth factor receptor (EGFR). However, the functional significance of Y211-phosphorylated PCNA remains unknown. Here, we show that PCNA phosphorylation by EGFR alters its interaction with mismatch-recognition proteins MutSα and MutSβ and interferes with PCNA-dependent activation of MutLα endonuclease, thereby inhibiting MMR at the initiation step. Evidence is also provided that Y211-phosphorylated PCNA induces nucleotide misincorporation during DNA synthesis. These findings reveal a novel mechanism by which Y211-phosphorylated PCNA promotes cancer development and progression via facilitating error-prone DNA replication and suppressing the MMR function.DNA mismatch repair (MMR) is an essential genome stability pathway that removes mismatches introduced by nucleotide misincorporation during DNA replication (1–4). MMR in eukaryotic cells is nick-directed and targeted specifically to the newly synthesized DNA strand (5, 6). MMR is carried out in three phases: initiation, excision, and resynthesis. The initiation phase involves mismatch recognition by MutSα or MutSβ; assembly of the initiation complex containing MutSα (or MutSβ), MutLα, and proliferating cell nuclear antigen (PCNA); and localization of the strand discrimination signal (a single-strand nick) by this complex (7–11) in a process that is incompletely understood. During the excision phase, exonuclease 1 (Exo1) removes nascent DNA exonucleolytically from a distal nick to the mismatch in a reaction that requires MutSα (or MutSβ), MutLα, and PCNA. Replication protein A (RPA) stimulates DNA excision by Exo1 and protects single-stranded DNA from cleavage by nucleases (12, 13). During the resynthesis phase of MMR, DNA polymerase δ fills in the single-strand DNA gap left by DNA excision in a concerted reaction that requires replication factor C (RFC), PCNA, and RPA, followed by DNA ligase I-catalyzed nick ligation.Mutations or promoter hypermethylation in coding or regulatory regions of MMR genes MSH2, MSH6, MLH1, and PMS2 are linked to susceptibility to colorectal and other cancers in humans and other organisms (2, 14–16). The cellular phenotype associated with defects in MMR includes an elevated genome-wide mutation rate as well as frequent changes in DNA microsatellite repeats, a phenomenon called microsatellite instability (MSI). Thus, MSI is frequently used as a biomarker for (or hallmark of) MMR deficiency (1–4). However, a significant number of MSI-positive tumors do not carry mutations in known MMR genes (17). Recent studies have shown that mutations affecting the proofreading nuclease activity of DNA polymerases δ and ε are associated with some colorectal and/or endometrial cancers (18), and that defects in the gene encoding histone H3 Lys36 (H3K36) trimethyltransferase SETD2 result in MSI and loss of MMR function in vivo (19).Although defects in MMR cause MSI and susceptibility to colorectal cancer, MSI-positive colorectal cancer cells are also closely associated with high levels of epidermal growth factor receptor (EGFR) (20), a transmembrane receptor protein kinase that promotes cell growth, tumor progression, and metastasis (21). EGFR overexpression in these cancer cells is mainly a result of its mRNA stability, which is caused by a one- or two-base deletion mutation within the A13/A14 repeat sequence in the 3′-untranslated region of the EGFR gene (20). EGFR can translocate to the nucleus (22) and phosphorylate PCNA on tyrosine 211 (Y211) to stimulate DNA synthesis and cell proliferation (23). Given that PCNA is required for the initiation and resynthesis steps of MMR (7, 9, 10), we hypothesized that EGFR-catalyzed PCNA phosphorylation might interfere with its role in MMR, leading to a mutator phenotype that promotes tumor progression.In this study, we compared the MMR activities of PCNA and Y211-phosphorylated PCNA (PCNA-Y211p) and examined the correlation between expression of EGFR with abundance of PCNA-Y211p and MMR activity in several human cancer cell lines. Our results suggest that a high level of PCNA-Y211p inhibits the initiation step of MMR and that this inhibition is reversed by excess exogenous nonphosphorylated PCNA. Cells expressing a high level of PCNA-Y211p display significantly reduced fidelity of DNA synthesis because of an elevated rate of nucleotide misincorporation. The results support the conclusion that phosphorylation of PCNA on Y211 inhibits MMR activity and reduces the fidelity of DNA synthesis. This may contribute to the mechanism by which EGFR family tyrosine kinases promote tumor progression.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.Heterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1–6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycine- and serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8–10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11, 12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12–25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12–20), mouse embryonic fibroblasts derived from Alk2^R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2^R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22, 26). In addition, despite the essential role of BMP signaling in development (27–31), the pre- and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1–6). These observations led us to hypothesize that FOP-ACVR1 abnormally responds to noncanonical BMP ligands induced by trauma or inflammation.Here we show that FOP-ACVR1 transduced BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling (10, 32–34) and contributes to inflammatory responses (35, 36). Our in vitro and in vivo data indicate that activation of TGF-β and aberrant BMP signaling by Activin-A in FOP-cells is one cause of HO in FOP. These results suggest a possible application of anti–Activin-A reagents as a new therapeutic tool for FOP.     

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.     

Lysine 63-linked polyubiquitination is required for EGF receptor degradation

Ubiquitination mediates endocytosis and endosomal sorting of various signaling receptors, transporters, and channels. However, the relative importance of mono- versus polyubiquitination and the role of specific types of polyubiquitin linkages in endocytic trafficking remain controversial. We used mass spectrometry-based targeted proteomics to show that activated epidermal growth factor receptor (EGFR) is ubiquitinated by one to two short (two to three ubiquitins) polyubiquitin chains mainly linked via lysine 63 (K63) or conjugated with a single monoubiquitin. Multimonoubiquitinated EGFR species were not found. To directly test whether K63 polyubiquitination is necessary for endocytosis and post-endocytic sorting of EGFR, a chimeric protein, in which the K63 linkage-specific deubiquitination enzyme AMSH [associated molecule with the Src homology 3 domain of signal transducing adaptor molecule (STAM)] was fused to the carboxyl terminus of EGFR, was generated. MS analysis of EGFR-AMSH ubiquitination demonstrated that the fraction of K63 linkages was substantially reduced, whereas relative amounts of monoubiquitin and K48 linkages increased, compared with that of wild-type EGFR. EGFR-AMSH was efficiently internalized into early endosomes, but, importantly, the rates of ligand-induced sorting to late endosomes and degradation of EGFR-AMSH were dramatically decreased. The slow degradation of EGFR-AMSH resulted in the sustained signaling activity of this chimeric receptor. Ubiquitination patterns, rate of endosomal sorting, and signaling kinetics of EGFR fused with the catalytically inactive mutant of AMSH were reversed to normal. Altogether, the data are consistent with the model whereby short K63-linked polyubiquitin chains but not multimonoubiquitin provide an increased avidity for EGFR interactions with ubiquitin adaptors, thus allowing rapid sorting of activated EGFR to the lysosomal degradation pathway.Ubiquitination, a posttranslational modification of proteins by attachment of the ubiquitin (Ub) polypeptide, is an important molecular signal that regulates endocytosis and post-endocytic sorting of membrane proteins (1–3). Ubiquitination is carried out by the sequential activity of E1, E2, and E3 enzymes; the latter, E3 ligases, typically determine the substrate specificity of Ub conjugation (4). Deubiquitinating enzymes (DUBs), a group of proteases capable of cleaving Ub from conjugates with target proteins, counteract the activity of the ubiquitination system (5). Ub is predominantly conjugated to lysine residues and much more rarely to the amino-terminal methionine or other amino acids in the substrate. Lysines and the amino-terminal methionine in the Ub molecule can also be conjugated to another molecule of Ub, leading to the formation of polyUb chains (6). Depending on the specific residue that links Ubs into a chain, polyUb chains have different molecular folding, are recognized by specific Ub-binding domains (UBDs) and have distinct functions (7). The structure and interaction mechanisms of lysine 48 (K48)- and K63-linked chains are most well-characterized (8–12). Crystal and NMR structures of K63 di-Ubs revealed extended, open conformation of two Ubs with high conformational freedom, as opposed to closed conformation of K48-polyUb linkages (reviewed in ref. 11). Therefore, ubiquitination substrates including endocytic cargo can be mono- and polyubiquitinated by different chains, but the role of these diverse types of ubiquitination in the regulation of endocytic trafficking remains incompletely understood.Epidermal growth factor (EGF) receptor (EGFR) was one of the first endocytic cargos in mammalian cells that were found to be ubiquitinated (13). This receptor has the profound role in eukaryotic development, regulation of various tissues in adult organisms, and pathogenesis of cancer (14). Therefore, EGFR has been a prototypic model for studying the mechanisms of endocytosis and endocytosis-relevant ubiquitination. EGFR is ubiquitinated by Cbl E3 ligases at the cell surface and after internalization in endosomes (15–17). The internalization step of EGFR trafficking is regulated by multiple redundant mechanisms, including ubiquitination, and is not significantly inhibited in the absence of receptor ubiquitination (18). By contrast, sorting of the internalized receptor in multivesicular bodies (MVBs), which leads to its incorporation into intraluminal vesicles of MVB and degradation in lysosomes, is highly sensitive to the extent of EGFR ubiquitination (15, 19).Based on differential recognition by Ub antibodies, EGFR was proposed to be conjugated with multiple monoUbs (20). Moreover, replacement of the cytoplasmic domain of EGFR with the Ub mutant incapable of polyubiquitination resulted in EGF-independent endocytosis and degradation of such chimeric receptor, thus suggesting that monoubiquitination is sufficient for EGFR endocytosis and MVB sorting (20). Subsequently, mass spectrometric (MS) analysis demonstrated a significant amount of EGFR polyubiquitination, mainly by K63-linked chains (19, 21, 22). However, whether K63 polyubiquitination is necessary for EGFR endocytic trafficking remains unknown.The role of cargo ubiquitination by K63-linked chains has been proposed in studies of endocytosis and MVB sorting of yeast permeases (23–27). These studies, however, used an approach of global elimination of K63 polyubiquitination in the cell to demonstrate the importance of these chains in endocytic trafficking. Because numerous proteins, including ESCRT components mediating MVB sorting are polyubiquitinated with K63 linkages, the inhibitory effects of the blockade of K63-linked polyubiquitination on endocytosis and MVB sorting observed in these studies may be indirect [e.g., not related to cargo ubiquitination (25)]. By contrast, an alternative approach based on the analysis of genetically engineered chimeric cargo molecules fused to Ub or a DUB demonstrated that monoubiquitination is fully sufficient for endocytosis and sorting of several membrane proteins to the vacuole in yeast (28).A number of mammalian endocytic cargo is polyubiquitinated by K63-linked chains (29–31) and, to a lesser extent, with K48 linkages (32–35). Similarly to studies in yeast, the role of these Ub linkages in mammalian cells was mainly examined by overexpressing K63R or K48R Ub mutants incapable of forming corresponding polyUb chains, leading to inhibition of K63 or K48 polyubiquitination of all cellular substrates (31–33). To test whether K63 polyubiquitination is required for EGFR endocytosis and endosomal sorting, we analyzed the stoichiometry of EGFR ubiquitination by MS and generated a chimeric EGFR fused at the carboxyl terminus to a DUB with the specificity toward K63 linkages. Analysis of the internalization and post-endocytic sorting of this chimeric receptor showed that K63 polyUb chains are necessary for the efficient EGF-induced down-regulation of EGFR.