PKCη regulates occludin phosphorylation and epithelial tight junction integrity

PKCη is expressed predominantly in the epithelial tissues; however, its role in the regulation of epithelial tight junctions (TJs) is unknown. We present evidence that PKCη phosphorylates occludin on threonine residues (T403 and T404) and plays a crucial role in the assembly and/or maintenance of TJs in Caco-2 and MDCK cell monolayers. Inhibition of PKCη by specific pseudo substrate inhibitor or knockdown of PKCη by specific shRNA disrupts the junctional distribution of occludin and ZO-1 and compromises the epithelial barrier function. Expression of dominant negative, PKCη_K394R disrupts the TJ and barrier function, whereas wild-type PKCη and constitutively active PKCη_A161E enhance the TJ integrity. Inhibition and knockdown of PKCη or expression of PKCη_K394R induce dephosphorylation of occludin on threonine residues, whereas active PKCη elevates occludin phosphorylation. PKCη directly interacts with the C-terminal domain of occludin and phosphorylates it on highly conserved T403 and T404. T403/404A mutations result in the loss of occludin''s ability to localize at the TJs, whereas T403/404D mutations attenuates the PKCη inhibitor-mediated redistribution of occludin from the intercellular junctions. These results reveal an important mechanism of epithelial TJ regulation by PKCη.     

PMLRARα binds to Fas and suppresses Fas-mediated apoptosis through recruiting c-FLIP in vivo

Defective Fas signaling leads to resistance to various anticancer therapies. Presence of potential inhibitors of Fas which could block Fas signaling can explain cancer cells resistance to apoptosis. We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PML-retinoic acid receptor α (PMLRARα). We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)-derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIP(L/S) and excluded procaspase 8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P < .001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Taken together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. The presence of PML-Fas complexes across different tissues implicates that PML functions in apoptosis regulation and tumor suppression are mediated by direct interaction with Fas.     

Wnt/β-catenin signaling is differentially regulated by Gα proteins and contributes to fibrous dysplasia

Skeletal dysplasias are common disabling disorders characterized by aberrant growth of bone and cartilage leading to abnormal skeletal structures and functions, often attributable to defects in skeletal progenitor cells. The underlying molecular and cellular mechanisms of most skeletal dysplasias remain elusive. Although the Wnt/β-catenin signaling pathway is required for skeletal progenitor cells to differentiate along the osteoblastic lineage, inappropriately elevated levels of signaling can also inhibit bone formation by suppressing osteoblast maturation. Here, we investigate interactions of the four major Gα protein families (Gα(s), Gα(i/o), Gα(q/11), and Gα(12/13)) with the Wnt/β-catenin signaling pathway and identify a causative role of Wnt/β-catenin signaling in fibrous dysplasia (FD) of bone, a disease that exhibits abnormal differentiation of skeletal progenitor cells. The activating Gα(s) mutations that cause FD potentiated Wnt/β-catenin signaling, and removal of Gα(s) led to reduced Wnt/β-catenin signaling and decreased bone formation. We further show that activation of Wnt/β-catenin signaling in osteoblast progenitors results in an FD-like phenotype and reduction of β-catenin levels rescued differentiation defects of FD patient-derived stromal cells. Gα proteins may act at the level of β-catenin destruction complex assembly by binding Axin. Our results indicate that activated Gα proteins differentially regulate Wnt/β-catenin signaling but, importantly, are not required core components of Wnt/β-catenin signaling. Our data suggest that activated Gα proteins are playing physiologically significant roles during both skeletal development and disease by modulating Wnt/β-catenin signaling strength.     

Setdb2 restricts dorsal organizer territory and regulates left–right asymmetry through suppressing fgf8 activity

Dorsal organizer formation is one of the most critical steps in early embryonic development. Several genes and signaling pathways that positively regulate the dorsal organizer development have been identified; however, little is known about the factor(s) that negatively regulates the organizer formation. Here, we show that Setdb2, a SET domain-containing protein possessing potential histone H3K9 methyltransferase activity, restricts dorsal organizer development and regulates left–right asymmetry by suppressing fibroblast growth factor 8 (fgf8) expression. Knockdown of Setdb2 results in a massive expansion of dorsal organizer markers floating head (flh), goosecoid (gsc), and chordin (chd), as well as a significant increase of fgf8, but not fgf4 mRNAs. Consequently, disrupted midline patterning and resultant randomization of left–right asymmetry are observed in Setdb2-deficient embryos. These characteristic changes induced by Setdb2 deficiency can be nearly corrected by either overexpression of a dominant-negative fgf receptor or knockdown of fgf8, suggesting an essential role for Setdb2–Fgf8 signaling in restricting dorsal organizer territory and regulating left–right asymmetry. These results provide unique evidence that a SET domain-containing protein potentially involved in the epigenetic control negatively regulates dorsal organizer formation during early embryonic development.     

DNMT3a epigenetic program regulates the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia

Epigenetic regulation of gene expression by DNA methylation plays a central role in the maintenance of cellular homeostasis. Here we present evidence implicating the DNA methylation program in the regulation of hypoxia-inducible factor (HIF) oxygen-sensing machinery and hypoxic cell metabolism. We show that DNA methyltransferase 3a (DNMT3a) methylates and silences the HIF-2α gene (EPAS1) in differentiated cells. Epigenetic silencing of EPAS1 prevents activation of the HIF-2α gene program associated with hypoxic cell growth, thereby limiting the proliferative capacity of adult cells under low oxygen tension. Naturally occurring defects in DNMT3a, observed in primary tumors and malignant cells, cause the unscheduled activation of EPAS1 in early dysplastic foci. This enables incipient cancer cells to exploit the HIF-2α pathway in the hypoxic tumor microenvironment necessary for the formation of cellular masses larger than the oxygen diffusion limit. Reintroduction of DNMT3a in DNMT3a-defective cells restores EPAS1 epigenetic silencing, prevents hypoxic cell growth, and suppresses tumorigenesis. These data support a tumor-suppressive role for DNMT3a as an epigenetic regulator of the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia.Metazoan life is dependent upon the use of molecular oxygen for an array of metabolic processes. Tissue hypoxia occurs during periods of imbalance between oxygen supply and consumption. One of the primary cellular responses to hypoxia is the activation of the hypoxia-inducible factor (HIF) program (1–4). HIF consists of oxygen-regulated α-subunits HIF-1α and HIF-2α and a constitutively expressed β-subunit (HIF-β). In the presence of oxygen, a series of nonheme Fe(II)- and 2-oxoglutarate–dependent dioxygenase oxygen sensors, referred to as HIF prolylhydroxylases (HIF PHDs), promote the hydroxylation of key proline residues on the HIF-α subunits (5, 6). This serves as a recognition site for the von Hippel-Lindau (VHL) tumor-suppressor protein, which mediates ubiquitination and proteasomal degradation of HIF-1α and HIF-2α (7–9). Hypoxia inhibits HIF PHDs, allowing HIF-1α and HIF-2α to evade VHL recognition and assemble with HIF-β to produce the active heterodimeric HIF factor. Once activated, HIF-1α and HIF-2α cooperate through common and distinct pathways to regulate hypoxic gene expression and cellular adaptation to hypoxia (10).A notable feature of the HIF response is the differential expression pattern of HIF-1α and HIF-2α in normal tissues. HIF-1α mRNA is ubiquitous and constitutively expressed in adult cells. In stark contrast, HIF-2α mRNA is detected in a few cell types of adult tissues and is typically not expressed by epithelia (11). This suggests a physiological necessity to fine-tune the HIF program depending upon the cellular settings by negatively regulating the HIF-2α gene (EPAS1) upstream of the HIF oxygen-sensing enzymes. The negative regulation of EPAS1 is often compromised in cancers, as HIF-2α mRNA is observed in the vast majority of overt tumors (11–13). This is particularly evident in renal cancer. Elegant studies by the Maxwell group (13) and others (14) revealed that HIF-2α mRNA is absent in human kidney tubule epithelia but present in dysplastic foci of the nephron. In these incipient renal tumor cells, HIF-2α may function as an oncoprotein (15), collaborating with, or activating, multiple growth-promoting pathways including cancer stewards c-myc (16), ras (17), and EGFR (18, 19). Silencing of HIF-2α suppresses tumorigenesis of various genetically diverse cancers, further highlighting its central role in malignancy (16, 17, 20, 21), although this depends on the experimental context (22). Therefore, EPAS1 is silent in adult epithelia but undergoes unscheduled activation in several malignancies, driving proliferation in the hypoxic tumor microenvironment (23).A clue to the mechanisms involved in the unscheduled activation of EPAS1 during early tumorigenesis may reside in its promoter, which harbors an enrichment of cytosine and guanine bases that often serve as sites of DNA methylation and epigenetic gene silencing (24–27). Cytosine methylation is catalyzed by a family of DNA methyltransferases (DNMTs) including DNMT1, DNMT3a, and DNMT3b. DNMT1 maintains the methylation pattern from the template strand to the newly synthesized strand during DNA replication (28). DNMT3a and DNMT3b are de novo methyltransferases that establish postreplicative methylation patterns (29). Alterations in DNA methylation patterns are common in tumors and likely play a central role in aberrant gene expression that characterizes the malignant phenotype (26, 30, 31). This is particularly evident for DNMT3a, as recent studies have identified mutations in DNMT3a in patients with acute myeloid leukemia (32, 33) or down-regulation of DNMT3a mRNA in a variety of solid tumors (34). It is suggested that DNMT3a is a tumor-suppressor gene and that its mutation, or mRNA down-regulation, contributes to reducing global DNMT3a methyltransferase activity (35, 36). Currently, a key challenge is to link aberrant methylation profiles commonly observed in malignant lesions, including alterations in the DNMT3a epigenetic program, to genes that directly promote the tumorigenic phenotype.Here we show that DNMT3a methylates and silences EPAS1 in normal cells. Loss of DNMT3a observed in primary tumors and malignant cells causes unscheduled EPAS1 activation. This allows emerging cancer cells to exploit the HIF-2α program that facilitates cancer cell traverse of the hypoxic barrier and formation of tumors larger than the diffusion limit of oxygen. We suggest that the DNMT3a epigenetic program is a gatekeeper of the hypoxic cancer cell phenotype.     

CaMKIIδ mediates β-adrenergic effects on RyR2 phosphorylation and SR Ca2 + leak and the pathophysiological response to chronic β-adrenergic stimulation

Chronic activation of Ca^2 +/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the deleterious effects of β-adrenergic receptor (β-AR) signaling on the heart, in part, by enhancing RyR2-mediated sarcoplasmic reticulum (SR) Ca^2 + leak. We used CaMKIIδ knockout (CaMKIIδ-KO) mice and knock-in mice with an inactivated CaMKII site S2814 on the ryanodine receptor type 2 (S2814A) to investigate the involvement of these processes in β-AR signaling and cardiac remodeling. Langendorff-perfused hearts from CaMKIIδ-KO mice showed inotropic and chronotropic responses to isoproterenol (ISO) that were similar to those of wild type (WT) mice; however, in CaMKIIδ-KO mice, CaMKII phosphorylation of phospholamban and RyR2 was decreased and isolated myocytes from CaMKIIδ-KO mice had reduced SR Ca^2 + leak in response to isoproterenol (ISO). Chronic catecholamine stress with ISO induced comparable increases in relative heart weight and other measures of hypertrophy from day 9 through week 4 in WT and CaMKIIδ-KO mice, but the development of cardiac fibrosis was prevented in CaMKIIδ-KO animals. A 4-week challenge with ISO resulted in reduced cardiac function and pulmonary congestion in WT, but not in CaMKIIδ-KO or S2814A mice, implicating CaMKIIδ-dependent phosphorylation of RyR2-S2814 in the cardiomyopathy, independent of hypertrophy, induced by prolonged β-AR stimulation.     

Role of CaMKIIδ phosphorylation of the cardiac ryanodine receptor in the force frequency relationship and heart failure

The force frequency relationship (FFR), first described by Bowditch 139 years ago as the observation that myocardial contractility increases proportionally with increasing heart rate, is an important mediator of enhanced cardiac output during exercise. Individuals with heart failure have defective positive FFR that impairs their cardiac function in response to stress, and the degree of positive FFR deficiency correlates with heart failure progression. We have identified a mechanism for FFR involving heart rate dependent phosphorylation of the major cardiac sarcoplasmic reticulum calcium release channel/ryanodine receptor (RyR2), at Ser2814, by calcium/calmodulin–dependent serine/threonine kinase–δ (CaMKIIδ). Mice engineered with an RyR2-S2814A mutation have RyR2 channels that cannot be phosphorylated by CaMKIIδ, and exhibit a blunted positive FFR. Ex vivo hearts from RyR2-S2814A mice also have blunted positive FFR, and cardiomyocytes isolated from the RyR2-S2814A mice exhibit impaired rate-dependent enhancement of cytosolic calcium levels and fractional shortening. The cardiac RyR2 macromolecular complexes isolated from murine and human failing hearts have reduced CaMKIIδ levels. These data indicate that CaMKIIδ phosphorylation of RyR2 plays an important role in mediating positive FFR in the heart, and that defective regulation of RyR2 by CaMKIIδ-mediated phosphorylation is associated with the loss of positive FFR in failing hearts.     

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.