Endoplasmic reticulum-associated degradation regulates mitochondrial dynamics in brown adipocytes.

The endoplasmic reticulum (ER) engages mitochondria at specialized ER domains known as mitochondria-associated membranes (MAMs). Here, we used three-dimensional high-resolution imaging to investigate the formation of pleomorphic "megamitochondria" with altered MAMs in brown adipocytes lacking the Sel1L-Hrd1 protein complex of ER-associated protein degradation (ERAD). Mice with ERAD deficiency in brown adipocytes were cold sensitive and exhibited mitochondrial dysfunction. ERAD deficiency affected ER-mitochondria contacts and mitochondrial dynamics, at least in part, by regulating the turnover of the MAM protein, sigma receptor 1 (SigmaR1). Thus, our study provides molecular insights into ER-mitochondrial cross-talk and expands our understanding of the physiological importance of Sel1L-Hrd1 ERAD.

PERK and XBP1 differentially regulate CXCL10 and CCL2 production.

Inflammation plays a key role in the pathogenesis of many retinal degenerative diseases related with photoreceptor dysfunction/degeneration. However the involvement of photoreceptor cells in inflammatory reactions is largely unknown as they are not considered as inflammatory cells. In this study, we assessed whether photoreceptor cells can produce CCL2 and CXCL10, two important players in inflammation during endoplasmic reticulum (ER) stress. After photoreceptor 661 W cells were treated with ER stress inducer thapsigargin (TG), induction of ER stress increased CXCL10 and CCL2 expression at both mRNA and protein levels, which was significantly blocked by an ER stress blocker 4-phenylbutyrate. ER stress contains three pathways: PERK, ATF6 and IRE1α. Knockdown of PERK attenuated TG-induced CXCL10 and CCL2 mRNA expression, associated with significant decreases in phosphorylation of NF-κB RelA and STAT3. In contrast to PERK, knockdown of XBP1, which is activated by IRE1α-mediated splicing, robustly enhanced TG-induced CXCL10 and CCL2 expression and phosphorylation of NF-κB RelA and STAT3. Blockade of NF-κB or STAT3 markedly diminished TG-induced CXCL10 and CCL2 expression. The specific roles of PERK and XBP1 in CXCL10 and CCL2 expression were further investigated by treating photoreceptor cells with advanced glycation end products (AGE) and high glucose (HG), two of the major contributors to diabetic complications. Similarly, AGE and HG induced CXCL10 and CCL2 expression in which PERK was a positive regulator while XBP1 was a negative regulator. These studies suggest that photoreceptors may be involved in retinal inflammation by expressing chemokines CXCL10 and CCL2. PERK and IRE1α/XBP1 in the unfolded protein response differentially regulate the expression of CXCL10 and CCL2 likely through modulation of ER stress-induced NF-κB RelA and STAT3 activation.

The Sel1L-Hrd1 Endoplasmic Reticulum-Associated Degradation Complex Manages a Key Checkpoint in B Cell Development.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a principal mechanism that targets ER-associated proteins for cytosolic proteasomal degradation. Here, our data demonstrate a critical role for the Sel1L-Hrd1 complex, the most conserved branch of ERAD, in early B cell development. Loss of Sel1L-Hrd1 ERAD in B cell precursors leads to a severe developmental block at the transition from large to small pre-B cells. Mechanistically, we show that Sel1L-Hrd1 ERAD selectively recognizes and targets the pre-B cell receptor (pre-BCR) for proteasomal degradation in a BiP-dependent manner. The pre-BCR complex accumulates both intracellularly and at the cell surface in Sel1L-deficient pre-B cells, leading to persistent pre-BCR signaling and pre-B cell proliferation. This study thus implicates ERAD mediated by Sel1L-Hrd1 as a key regulator of B cell development and reveals the molecular mechanism underpinning the transient nature of pre-BCR signaling.

IRE1α is an endogenous substrate of endoplasmic-reticulum-associated degradation.

Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however, its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of the unfolded protein response (UPR), is a bona fide substrate of the Sel1L-Hrd1 ERAD complex. ERAD-mediated IRE1α degradation occurs under basal conditions in a BiP-dependent manner, requires both the intramembrane hydrophilic residues of IRE1α and the lectin protein OS9, and is attenuated by ER stress. ERAD deficiency causes IRE1α protein stabilization, accumulation and mild activation both in vitro and in vivo. Although enterocyte-specific Sel1L-knockout mice (Sel1L(ΔIEC)) are viable and seem normal, they are highly susceptible to experimental colitis and inflammation-associated dysbiosis, in an IRE1α-dependent but CHOP-independent manner. Hence, Sel1L-Hrd1 ERAD serves a distinct, essential function in restraint of IRE1α signalling in vivo by managing its protein turnover.

The ER-associated degradation adaptor protein Sel1L regulates LPL secretion and lipid metabolism.

Sel1L is an essential adaptor protein for the E3 ligase Hrd1 in the endoplasmic reticulum (ER)-associated degradation (ERAD), a universal quality-control system in the cell; but its physiological role remains unclear. Here we show that mice with adipocyte-specific Sel1L deficiency are resistant to diet-induced obesity and exhibit postprandial hypertriglyceridemia. Further analyses reveal that Sel1L is indispensable for the secretion of lipoprotein lipase (LPL), independent of its role in Hrd1-mediated ERAD and ER homeostasis. Sel1L physically interacts with and stabilizes the LPL maturation complex consisting of LPL and lipase maturation factor 1 (LMF1). In the absence of Sel1L, LPL is retained in the ER and forms protein aggregates, which are degraded primarily by autophagy. The Sel1L-mediated control of LPL secretion is also seen in other LPL-expressing cell types including cardiac myocytes and macrophages. Thus, our study reports a role of Sel1L in LPL secretion and systemic lipid metabolism.

Adipocyte spliced form of X-box-binding protein 1 promotes adiponectin multimerization and systemic glucose homeostasis.

The physiological role of the spliced form of X-box-binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in adipocytes, thereby regulating systemic glucose homeostasis. Ectopic expression of XBP1s in adipocytes improves glucose tolerance and insulin sensitivity in both lean and obese (ob/ob) mice. The beneficial effect of adipocyte XBP1s on glucose homeostasis is associated with elevated serum levels of high-molecular-weight adiponectin and, indeed, is adiponectin-dependent. Mechanistically, XBP1s promotes adiponectin multimerization rather than activating its transcription, likely through a direct regulation of the expression of several ER chaperones involved in adiponectin maturation, including glucose-regulated protein 78 kDa, protein disulfide isomerase family A, member 6, ER protein 44, and disulfide bond oxidoreductase A-like protein. Thus, we conclude that XBP1s is an important regulator of adiponectin multimerization, which may lead to a new therapeutic approach for the treatment of type 2 diabetes and hypoadiponectinemia.

Phenformin activates the unfolded protein response in an AMP-activated protein kinase (AMPK)-dependent manner.

BACKGROUND: The cross-talk between UPR activation and metabolic stress remains largely unclear. RESULTS: Phenformin treatment activates the IRE1α and PERK pathways in an AMPK-dependent manner. CONCLUSION: AMPK is required for phenformin-mediated IRE1α and PERK activation. SIGNIFICANCE: Our findings demonstrate the cross-talk between UPR and metabolic signals. Activation of the unfolded protein response (UPR) is associated with the disruption of endoplasmic reticulum (ER) homeostasis and has been implicated in the pathogenesis of many human metabolic diseases, including obesity and type 2 diabetes. However, the nature of the signals activating UPR under these conditions remains largely unknown. Using a method that we recently optimized to directly measure UPR sensor activation, we screened the effect of various metabolic drugs on UPR activation and show that the anti-diabetic drug phenformin activates UPR sensors IRE1α and pancreatic endoplasmic reticulum kinase (PERK) in both an ER-dependent and ER-independent manner. Mechanistically, AMP-activated protein kinase (AMPK) activation is required but not sufficient to initiate phenformin-mediated IRE1α and PERK activation, suggesting the involvement of additional factor(s). Interestingly, activation of the IRE1α (but not PERK) pathway is partially responsible for the cytotoxic effect of phenformin. Together, our data show the existence of a non-canonical UPR whose activation requires the cytosolic kinase AMPK, adding another layer of complexity to UPR activation upon metabolic stress.

Fstl1 antagonizes BMP signaling and regulates ureter development.

Bone morphogenetic protein (BMP) signaling pathway plays important roles in urinary tract development although the detailed regulation of its activity in this process remains unclear. Here we report that follistatin-like 1 (Fstl1), encoding a secreted extracellular glycoprotein, is expressed in developing ureter and antagonizes BMP signaling activity. Mouse embryos carrying disrupted Fstl1 gene displayed prominent hydroureter arising from proximal segment and ureterovesical junction defects. These defects were associated with significant reduction in ureteric epithelial cell proliferation at E15.5 and E16.5 as well as absence of subepithelial ureteral mesenchymal cells in the urinary tract at E16.5 and E18.5. At the molecular level, increased BMP signaling was found in Fstl1 deficient ureters, indicated by elevated pSmad1/5/8 activity. In vitro study also indicated that Fstl1 can directly bind to ALK6 which is specifically expressed in ureteric epithelial cells in developing ureter. Furthermore, Sonic hedgehog (SHH) signaling, which is crucial for differentiation of ureteral subepithelial cell proliferation, was also impaired in Fstl1(-/-) ureter. Altogether, our data suggest that Fstl1 is essential in maintaining normal ureter development by antagonizing BMP signaling.

Stressed out about obesity: IRE1α-XBP1 in metabolic disorders.

The global obesity epidemic is associated with a series of health-threatening diseases including type 2 diabetes. Accumulating evidence suggest that the physiology and homeostasis of the endoplasmic reticulum (ER) is intimately involved in the underlying mechanisms linking obesity and diabetes. Specifically, recent studies indicate a crucial role for the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 (XBP1) pathway, the most conserved branch of the unfolded protein response (UPR), in glucose and lipid metabolism as well as in insulin function. Focusing on the IRE1α-XBP1 pathway, we review recent advances in our understanding of the role of UPR in obesity and obesity-associated metabolic disorders.

Gr-1+ CD11b+ myeloid-derived suppressor cells suppress inflammation and promote insulin sensitivity in obesity.

Activation of immune cells, including macrophages and CD8(+) T cells, contributes significantly to the advancement of obesity and its associated medical complications, such as atherosclerosis, insulin resistance, and type 2 diabetes. However, how the activation of these immune cells is regulated in vivo remains largely unexplored. Here we show that a group of immature myeloid cells with cell surface markers of Gr-1(+) CD11b(+) are highly enriched in peripheral tissues (i.e. liver and adipose tissues) during obesity. Down-regulation of these cells in obese animals significantly increases inflammation and impairs insulin sensitivity and glucose tolerance, whereas elevation of these cells via adoptive transfer has the opposite effects. Mechanistically, we show that under obese conditions, the Gr-1(+) cells suppress proliferation and induce apoptosis of CD8(+) T cells and are capable of skewing differentiation of macrophages into insulin-sensitizing, alternatively activated M2 macrophages. Taken together, our study demonstrates that immature myeloid cells provide a checks-and-balances platform to counter proinflammatory immune cells in the liver and adipose tissue during obesity to prevent overt immune responses.

Haploid insufficiency of suppressor enhancer Lin12 1-like (SEL1L) protein predisposes mice to high fat diet-induced hyperglycemia.

Increasing evidence suggests that endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of type 2 diabetes mellitus. SEL1L is an ER membrane protein that is highly expressed in the pancreatic islet and acinar cells. We have recently reported that a deficiency of SEL1L causes systemic ER stress and leads to embryonic lethality in mice. Here we show that mice with one functional allele of Sel1l (Sel1l(+/-)) are more susceptible to high fat diet (HFD)-induced hyperglycemia. Sel1l(+/-) mice have a markedly reduced ß-cell mass as a result of decreased ß-cell proliferation. Consequently, Sel1l(+/-) mice are severely glucose-intolerant and exhibit significantly retarded glucose-stimulated insulin secretion. Pancreatic islets from Sel1l(+/-) mice stimulated with a high concentration of glucose in vitro express significantly higher levels of unfolded protein response genes than those from wild-type control mice. Furthermore, dominant-negative interference of SEL1L function in insulinoma cell lines severely impairs, whereas overexpression of SEL1L efficiently improves protein secretion. Taken together, our results indicate that haploid insufficiency of SEL1L predispose mice to high fat diet-induced hyperglycemia. Our findings highlight a critical and previously unknown function for SEL1L in regulating adult ß-cell function and growth.

Emerging roles for XBP1, a sUPeR transcription factor.

X-box binding protein 1 (XBP1) is a unique basic region leucine zipper (bZIP) transcription factor whose active form is generated by a nonconventional splicing reaction upon disruption of homeostasis in the endoplasmic reticulum (ER) and activation of the unfolded protein response (UPR). XBP1, first identified as a key regulator of major histocompatibility complex (MHC) class II gene expression in B cells, represents the most conserved signaling component of UPR and is critical for cell fate determination in response to ER stress. Here we review recent advances in our understanding of this multifaceted transcription factor in health and diseases.

XBP-1 couples endoplasmic reticulum stress to augmented IFN-beta induction via a cis-acting enhancer in macrophages.

Perturbation of the endoplasmic reticulum (ER) results in a conserved stress response called the unfolded protein response (UPR). Macrophages undergoing a UPR respond to LPS with log-fold increased production of IFN-beta, a cytokine with diverse roles in innate and adaptive immunity. In this study, we found that thapsigargin-induced ER stress augmented recruitment of IFN regulatory factor-3, CREB binding protein/p300, and transcriptional machinery to the murine ifnb1 promoter during LPS stimulation. Although full synergistic IFN-beta production requires X-box binding protein 1 (XBP-1), this UPR-regulated transcription factor did not appreciably bind the ifnb1 promoter. However, XBP-1 bound a conserved site 6.1 kb downstream of ifnb1, along with IFN regulatory factor-3 and CREB binding protein only during concomitant UPR and LPS stimulation. XBP-1 physically associates with p300, suggesting a mechanism of multimolecular assembly at the +6.1 kb site. Luciferase reporter assays provide evidence this +6 kb region functions as an XBP-1-dependent enhancer of ifnb1 promoter activity. Thus, this study identifies a novel role for a UPR-dependent transcription factor in the regulation of an inflammatory cytokine. Our findings have broader mechanistic implications for the pathogenesis of diseases involving ER stress and type I IFN, including viral infection, ischemia-reperfusion injury, protein misfolding, and inflammatory diseases.

The IRE1alpha-XBP1 pathway of the unfolded protein response is required for adipogenesis.

Signaling cascades during adipogenesis culminate in the expression of two essential adipogenic factors, PPARgamma and C/EBPalpha. Here we demonstrate that the IRE1alpha-XBP1 pathway, the most conserved branch of the unfolded protein response (UPR), is indispensable for adipogenesis. Indeed, XBP1-deficient mouse embryonic fibroblasts and 3T3-L1 cells with XBP1 or IRE1alpha knockdown exhibit profound defects in adipogenesis. Intriguingly, C/EBPbeta, a key early adipogenic factor, induces Xbp1 expression by directly binding to its proximal promoter region. Subsequently, XBP1 binds to the promoter of Cebpa and activates its gene expression. The posttranscriptional splicing of Xbp1 mRNA by IRE1alpha is required as only the spliced form of XBP1 (XBP1s) rescues the adipogenic defect exhibited by XBP1-deficient cells. Taken together, our data show that the IRE1alpha-XBP1 pathway plays a key role in adipocyte differentiation by acting as a critical regulator of the morphological and functional transformations during adipogenesis.

Disruption of a novel regulatory locus results in decreased Bdnf expression, obesity, and type 2 diabetes in mice.

Mutants of brain-derived neurotrophic factor (BDNF) are associated with obesity. However, the regulatory mechanism of BDNF expression is still unclear. We developed a novel mutant mouse line, transgenic insertional mutants with obesity, named Timo, in which a potential regulatory locus of Bdnf was disrupted by transgene insertion. The insertion site was identified and lies 857 kb upstream of the Bdnf gene. The disrupted genomic locus is conserved across the mouse, rat, dog, and human genome and contains several highly conserved elements that are able to upregulate reporter gene expression in vitro. Along with downregulation of BDNF to approximately 30% of wild-type animals, Timo/Timo mice exhibited increased body weight and fat content with hepatic steatosis and elevated serum levels of leptin, cholesterol, and LDL cholesterol. These mutant mice also showed obesity-independent insulin resistance, hyperinsulinemia, impaired glucose tolerance, age-dependent hyperglycemia, and shortened life span. Molecular and phenotype analysis of Timo/Timo mice indicated the existence of a genome locus, lying 857 kb upstream of the Bdnf gene, that regulates BDNF expression, body weight, and glucose homeostasis.

Alternative 3' UTR polyadenylation of Bzw1 transcripts display differential translation efficiency and tissue-specific expression.

BZW1 is a conserved regulatory factor for transcriptional control of histone H4 gene at the G1/S transition. In this study, three Bzw1 transcripts were identified in mice with two long forms (approximately 2.9 kb) expressed ubiquitously at low level, and a short transcript of 1.8 kb expressed at high level exclusively in testis. These different transcripts share the same 5' UTR and coding sequence, but differ in the length of 3' UTR by utilizing alternative polyadenylation sites. Different translation efficiencies were observed in the cells transfected with chimeric EGFP-Bzw1 genes tailed with different 3' UTRs. Our results demonstrate that Bzw1 transcripts are alternatively polyadenylated and expressed in tissue-specific pattern.

Phosphatase of regenerating liver-3 promotes motility and metastasis of mouse melanoma cells.

Recent reports suggested that phosphatase of regenerating liver (PRL)-3 might be involved in colorectal carcinoma metastasis with an unknown mechanism. Here we demonstrated that PRL-3 expression was up-regulated in human liver carcinoma compared with normal liver. PRL-3 was also highly expressed in metastatic melanoma B16-BL6 cells but not in its lowly metastatic parental cell line, B16 cells. B16 cells transfected with PRL-3 cDNA displayed morphological transformation from epithelial-like shape to fibroblast-like shape. PRL-3-overexpressed cells showed much higher migratory ability, which could be reversed by specific anti-sense oligodeoxynucleotide and the phosphatase inhibitors sodium orthovanadate or potassium bisperoxo oxovanadate V. Meanwhile, the expression of the catalytically inactive PRL-3 mutations (D72A or C104S) significantly reduced the cell migratory capability. In addition, PRL-3 transfectants demonstrated altered extracellular matrix adhesive property and up-regulated integrin-mediated cell spreading efficiency. Furthermore, we confirmed that PRL-3 could facilitate lung and liver metastasis of B16 cells in an experimental metastasis model in mice, consistent with accelerated proliferation and growth rate both in vitro and in vivo. Together, these observations provide convincing evidence that PRL-3 truly plays a causal role in tumor metastasis.