Hepatic Sel1L-Hrd1 ER-associated degradation (ERAD) manages FGF21 levels and systemic metabolism via CREBH.

Fibroblast growth factor 21 (Fgf21) is a liver-derived, fasting-induced hormone with broad effects on growth, nutrient metabolism, and insulin sensitivity. Here, we report the discovery of a novel mechanism regulating Fgf21 expression under growth and fasting-feeding. The Sel1L-Hrd1 complex is the most conserved branch of mammalian endoplasmic reticulum (ER)-associated degradation (ERAD) machinery. Mice with liver-specific deletion of Sel1L exhibit growth retardation with markedly elevated circulating Fgf21, reaching levels close to those in Fgf21 transgenic mice or pharmacological models. Mechanistically, we show that the Sel1L-Hrd1 ERAD complex controls Fgf21 transcription by regulating the ubiquitination and turnover (and thus nuclear abundance) of ER-resident transcription factor Crebh, while having no effect on the other well-known Fgf21 transcription factor Pparα. Our data reveal a physiologically regulated, inverse correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 levels under fasting-feeding and growth. This study not only establishes the importance of Sel1L-Hrd1 ERAD in the liver in the regulation of systemic energy metabolism, but also reveals a novel hepatic "ERAD-Crebh-Fgf21" axis directly linking ER protein turnover to gene transcription and systemic metabolic regulation.

ERAD deficiency promotes mitochondrial dysfunction and transcriptional rewiring in human hepatic cells.

Mitochondrial dysfunction is associated with a variety of human diseases including neurodegeneration, diabetes, nonalcohol fatty liver disease (NAFLD), and cancer, but its underlying causes are incompletely understood. Using the human hepatic cell line HepG2 as a model, we show here that endoplasmic reticulum-associated degradation (ERAD), an ER protein quality control process, is critically required for mitochondrial function in mammalian cells. Pharmacological inhibition or genetic ablation of key proteins involved in ERAD increased cell death under both basal conditions and in response to proinflammatory cytokines, a situation frequently found in NAFLD. Decreased viability of ERAD-deficient HepG2 cells was traced to impaired mitochondrial functions including reduced ATP production, enhanced reactive oxygen species (ROS) accumulation, and increased mitochondrial outer membrane permeability. Transcriptome profiling revealed widespread down-regulation of genes underpinning mitochondrial functions, and up-regulation of genes associated with tumor growth and aggression. These results highlight a critical role for ERAD in maintaining mitochondrial functional and structural integrity and raise the possibility of improving cellular and organismal mitochondrial function via enhancing cellular ERAD capacity.

Insm1 promotes endocrine cell differentiation by modulating the expression of a network of genes that includes Neurog3 and Ripply3.

Insulinoma associated 1 (Insm1) plays an important role in regulating the development of cells in the central and peripheral nervous systems, olfactory epithelium and endocrine pancreas. To better define the role of Insm1 in pancreatic endocrine cell development we generated mice with an Insm1(GFPCre) reporter allele and used them to study Insm1-expressing and null populations. Endocrine progenitor cells lacking Insm1 were less differentiated and exhibited broad defects in hormone production, cell proliferation and cell migration. Embryos lacking Insm1 contained greater amounts of a non-coding Neurog3 mRNA splice variant and had fewer Neurog3/Insm1 co-expressing progenitor cells, suggesting that Insm1 positively regulates Neurog3. Moreover, endocrine progenitor cells that express either high or low levels of Pdx1, and thus may be biased towards the formation of specific cell lineages, exhibited cell type-specific differences in the genes regulated by Insm1. Analysis of the function of Ripply3, an Insm1-regulated gene enriched in the Pdx1-high cell population, revealed that it negatively regulates the proliferation of early endocrine cells. Taken together, these findings indicate that in developing pancreatic endocrine cells Insm1 promotes the transition from a ductal progenitor to a committed endocrine cell by repressing a progenitor cell program and activating genes essential for RNA splicing, cell migration, controlled cellular proliferation, vasculogenesis, extracellular matrix and hormone secretion.

Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival.

Suppressor/Enhancer of Lin-12-like (Sel1L) is an adaptor protein for the E3 ligase hydroxymethylglutaryl reductase degradation protein 1 (Hrd1) involved in endoplasmic reticulum-associated degradation (ERAD). Sel1L's physiological importance in mammalian ERAD, however, remains to be established. Here, using the inducible Sel1L knockout mouse and cell models, we show that Sel1L is indispensable for Hrd1 stability, ER homeostasis, and survival. Acute loss of Sel1L leads to premature death in adult mice within 3 wk with profound pancreatic atrophy. Contrary to current belief, our data show that mammalian Sel1L is required for Hrd1 stability and ERAD function both in vitro and in vivo. Sel1L deficiency disturbs ER homeostasis, activates ER stress, attenuates translation, and promotes cell death. Serendipitously, using a biochemical approach coupled with mass spectrometry, we found that Sel1L deficiency causes the aggregation of both small and large ribosomal subunits. Thus, Sel1L is an indispensable component of the mammalian Hrd1 ERAD complex and ER homeostasis, which is essential for protein translation, pancreatic function, and cellular and organismal survival.

SEL1L preserves CD8+ T-cell survival and homeostasis by fine-tuning PERK signaling and the IL-15 receptor-mediated mTORC1 axis.

SEL1L-mediated endoplasmic reticulum-associated degradation (ERAD) plays critical roles in controlling protein homeostasis by degrading misfolded or terminal unfolded proteins. However, it remains unclear how SEL1L regulates peripheral T-cell survival and homeostasis. Herein, we found that SEL1L deficiency led to a greatly reduced frequency and number of mature T cells, which was further validated by adoptive transfer experiments or bone marrow chimera experiments, accompanied by the induction of multiple forms of cell death. Furthermore, SEL1L deficiency selectively disrupted naïve CD8+ T-cell homeostasis, as indicated by the severe loss of the naïve T-cell subset but an increase in the memory T-cell subset. We also found that SEL1L deficiency fueled mTORC1/c-MYC activation and induced a metabolic shift, which was largely attributable to enhanced expression of the IL-15 receptor α and ß chains. Mechanistically, single-cell transcriptomic profiling and biochemical analyses further revealed that Sel1l-/- CD8+ T cells harbored excessive ER stress, particularly aberrant activation of the PERK-ATF4-CHOP-Bim pathway, which was alleviated by supplementing IL-7 or IL-15. Importantly, PERK inhibition greatly resolved the survival defects of Sel1l-/- CD8+ T cells. In addition, IRE1α deficiency decreased mTORC1 signaling in Sel1l-/- naïve CD8+ T cells by downregulating the IL-15 receptor α chain. Altogether, these observations suggest that the ERAD adaptor molecule SEL1L acts as an important checkpoint for preserving the survival and homeostasis of peripheral T cells by regulating the PERK signaling cascade and IL-15 receptor-mediated mTORC1 axis.

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.

mSEL-1L (Suppressor/enhancer Lin12-like) protein levels influence murine neural stem cell self-renewal and lineage commitment.

Murine SEL-1L (mSEL-1L) is a key component of the endoplasmic reticulum-associated degradation pathway. It is essential during development as revealed by the multi-organ dysfunction and in uterus lethality occurring in homozygous mSEL-1L-deficient mice. Here we show that mSEL-1L is highly expressed in pluripotent embryonic stem cells and multipotent neural stem cells (NSCs) but silenced in all mature neural derivatives (i.e. astrocytes, oligodendrocytes, and neurons) by mmu-miR-183. NSCs derived from homozygous mSEL-1L-deficient embryos (mSEL-1L(-/-) NSCs) fail to proliferate in vitro, show a drastic reduction of the Notch effector HES-5, and reveal a significant down-modulation of the early neural progenitor markers PAX-6 and OLIG-2, when compared with the wild type (mSEL-1L(+/+) NSCs) counterpart. Furthermore, these cells are almost completely deprived of the neural marker Nestin, display a significant decrease of SOX-2 expression, and rapidly undergo premature astrocytic commitment and apoptosis. The data suggest severe self-renewal defects occurring in these cells probably mediated by misregulation of the Notch signaling. The results reported here denote mSEL-1L as a primitive marker with a possible involvement in the regulation of neural progenitor stemness maintenance and lineage determination.

Mechano growth factor (MGF) promotes proliferation and inhibits differentiation of porcine satellite cells (PSCs) by down-regulation of key myogenic transcriptional factors.

Porcine satellite cells represent an ideal model system for studying the cellular and molecular basis regulating myogenic stem cell proliferation and differentiation and for exploring the experimental conditions for myoblast transplantation. Here, we investigated the effects of mechano growth factor (MGF), a spliced variant of the IGF-1 gene, on porcine satellite cells. We show that MGF potently stimulated proliferation while inhibited differentiation of porcine satellite cells. MGF-treatment acutely down-regulates the expression of myogenic determination factor (MyoD) and the cyclin-dependent kinase inhibitor p21. MGF-treatment also markedly reduced the overall expression of cyclin B1 and key factors of the myogenic regulatory and myocyte enhancer families, including Myogenein and MEF2A. Taken together, the gene expression data from MGF-treated porcine satellite cells are in favor of a molecular model in which MGF inhibits porcine satellite cell differentiation by down-regulating either the activity or expression of MyoD, which, in turn, suppresses the expression of key genes required for cell cycle progression and differentiation, such as p21, Myogenin, and MEF2. Overall, our findings are in support of the previous suggestion that MGF may be used in vivo and in vitro to promote proliferation of myogenic stem cells to prevent and treat age-related muscle degenerative diseases.

T-cell-specific Sel1L deletion exacerbates EAE by promoting Th1/Th17-cell differentiation.

Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) are demyelinating neuroinflammatory diseases identified by the accumulation and aggregation of misfolded proteins in the brain. The Sel1L-Hrd1 complex comprising endoplasmic reticulum associated degradation (ERAD) is an ER-protein quality control system (ERQC) in the cell. Unfortunately, the contribution of ERAD to the development of these diseases has not been well explored. In this study, we used mice with a conditional deletion (KO) of Sel1L in T cells to dissect the role of ERAD on T cells and its contribution to the development of EAE. The results showed that Sel1L KO mice developed more severe EAE than the control wild type (WT) mice. Although, no obvious effects on peripheral T cells in steady state, more CD44-CD25+ double-negative stage 3 (DN3) cells were detected in the thymus. Moreover, Sel1L deficiency promoted the differentiation of Th1 and Th17 cells and upregulated the proliferation and apoptosis of CD4 T cells in vitro. Regarding the mechanism analyzed by RNA sequencing, 437 downregulated genes and 271 upregulated genes were detected in Sel1L deletion CD4 T cells, which covered the activation, proliferation, differentiation and apoptosis of these T cells. Thus, this study declared that the dysfunction of Sel1L in ERAD in T cells exacerbated the severity of EAE and indicated the important role of ERQC in maintaining immune homeostasis in the central nervous system.

Deficiency of suppressor enhancer Lin12 1 like (SEL1L) in mice leads to systemic endoplasmic reticulum stress and embryonic lethality.

Stress in the endoplasmic reticulum (ER) plays an important causal role in the pathogenesis of several chronic diseases such as Alzheimer, Parkinson, and diabetes mellitus. Insight into the genetic determinants responsible for ER homeostasis will greatly facilitate the development of therapeutic strategies for the treatment of these debilitating diseases. Suppressor enhancer Lin12 1 like (SEL1L) is an ER membrane protein and was thought to be involved in the quality control of secreted proteins. Here we show that the mice homozygous mutant for SEL1L were embryonic lethal. Electron microscopy studies revealed a severely dilated ER in the fetal liver of mutant embryos, indicative of alteration in ER homeostasis. Consistent with this, several ER stress responsive genes were significantly up-regulated in the mutant embryos. Mouse embryonic fibroblast cells deficient in SEL1L exhibited activated unfolded protein response at the basal state, impaired ER-associated protein degradation, and reduced protein secretion. Furthermore, markedly increased apoptosis was observed in the forebrain and dorsal root ganglions of mutant embryos. Taken together, our results demonstrate an essential role for SEL1L in protein quality control during mouse embryonic development.

Replacement of Rbpj with Rbpjl in the PTF1 complex controls the final maturation of pancreatic acinar cells.

BACKGROUND & AIMS: The mature pancreatic acinar cell is dedicated to the production of very large amounts of digestive enzymes. The early stages of pancreatic development require the Rbpj form of the trimeric Pancreas Transcription Factor 1 complex (PTF1-J). As acinar development commences, Rbpjl gradually replaces Rbpj; in the mature pancreas, PTF1 contains Rbpjl (PTF1-L). We investigated whether PTF1-L controls the expression of genes that complete the final stage of acinar differentiation. METHODS: We analyzed acinar development and transcription in mice with disrupted Rbpjl (Rbpjl(ko/ko) mice). We performed comprehensive analyses of the messenger RNA population and PTF1 target genes in pancreatic acinar cells from these and wild-type mice. RESULTS: In Rbpjl(ko/ko) mice, acinar differentiation was incomplete and characterized by decreased expression (as much as 99%) of genes that encode digestive enzymes or proteins of regulated exocytosis and mitochondrial metabolism. Whereas PTF1-L bound regulatory sites of genes in normal adult pancreatic cells, the embryonic form (PTF1-J) persisted in the absence of Rbpjl and replaced PTF1-L; the extent of replacement determined gene expression levels. Loss of PTF1-L reduced expression (>2-fold) of only about 50 genes, 90% of which were direct targets of PTF1-L. The magnitude of the effects on individual digestive enzyme genes correlated with the developmental timing of gene activation. Absence of Rbpjl increased pancreatic expression of liver-restricted messenger RNA. CONCLUSIONS: Replacement of Rbpj by Rbpjl in the PTF1 complex drives acinar differentiation by maximizing secretory protein synthesis, stimulating mitochondrial metabolism and cytoplasmic creatine-phosphate energy stores, completing the packaging and secretory apparatus, and maintaining acinar-cell homeostasis.

Reducing embryonic mtDNA copy number alters epigenetic profile of key hepatic lipolytic genes and causes abnormal lipid accumulation in adult mice.

Adverse fetal environment, in particular a shortage or excess of nutrients, is associated with increased risks of metabolic diseases later in life. However, the molecular mechanisms underlying this developmental origin of adult diseases remain unclear. Here, we directly tested the role of mitochondrial stress in mediating fetal programming in mice by enzymatically depleting mtDNA in zygotes. mtDNA-targeted plasmid microinjection is used to reduce embryonic mtDNA copy number directly, followed by embryo transfer. Mice with reduced zygote mtDNA copy number were born morphologically normal and showed no accelerated body weight gain. However, at 5 months of age these mice showed markedly increased hepatic lipidosis and became glucose-intolerant. Hepatic mRNA and protein expressions of peroxisome proliferator-activated receptor α (Pparα), a key transcriptional regulator of lipid metabolism, were significantly decreased as a result of increased DNA methylation in its proximal regulatory region. These results indicate that perturbation of mitochondrial function around the periconceptional period causes hypermethylation and thus suppressed expression of PPARα in fetal liver, leading to impaired hepatic lipid metabolism. Our findings provide the first direct evidence that mitochondrial stress mediates epigenetic changes associated with fetal programming of adult diseases in a mammalian system.

SEL1L deficiency impairs growth and differentiation of pancreatic epithelial cells.

BACKGROUND: The vertebrate pancreas contains islet, acinar and ductal cells. These cells derive from a transient pool of multipotent pancreatic progenitors during embryonic development. Insight into the genetic determinants regulating pancreatic organogenesis will help the development of cell-based therapies for the treatment of diabetes mellitus. Suppressor enhancer lin12/Notch 1 like (Sel1l) encodes a cytoplasmic protein that is highly expressed in the developing mouse pancreas. However, the morphological and molecular events regulated by Sel1l remain elusive. RESULTS: We have characterized the pancreatic phenotype of mice carrying a gene trap mutation in Sel1l. We show that Sel1l expression in the developing pancreas coincides with differentiation of the endocrine and exocrine lineages. Mice homozygous for the gene trap mutation die prenatally and display an impaired pancreatic epithelial morphology and cell differentiation. The pancreatic epithelial cells of Sel1l mutant embryos are confined to the progenitor cell state throughout the secondary transition. Pharmacological inhibition of Notch signaling partially rescues the pancreatic phenotype of Sel1l mutant embryos. CONCLUSIONS: Together, these data suggest that Sel1l is essential for the growth and differentiation of endoderm-derived pancreatic epithelial cells during mouse embryonic development.

Pdx-1 and Ptf1a concurrently determine fate specification of pancreatic multipotent progenitor cells.

The pancreas is derived from a pool of multipotent progenitor cells (MPCs) that co-express Pdx-1 and Ptf1a. To more precisely define how the individual and combined loss of Pdx-1 and Ptf1a affects pancreatic MPC specification and differentiation we derived and studied mice bearing a novel Ptf1a(YFP) allele. While the expression of Pdx-1 and Ptf1a in pancreatic MPCs coincides between E9.5 and 12.5 the developmental phenotypes of Pdx-1 null and Pdx-1; Ptf1a double null mice are indistinguishable, and an early pancreatic bud is formed in both cases. This finding indicates that Pdx-1 is required in the foregut endoderm prior to Ptf1a for pancreatic MPC specification. We also found that Ptf1a is neither required for specification of Ngn3-positive endocrine progenitors nor differentiation of mature beta-cells. In the absence of Pdx-1 Ngn3-positive cells were not observed after E9.5. Thus, in contrast to the deletion of Ptf1a, the loss of Pdx-1 precludes the sustained Ngn3-based derivation of endocrine progenitors from pancreatic MPCs. Taken together, these studies indicate that Pdx-1 and Ptf1a have distinct but interdependent functions during pancreatic MPC specification.

Endoplasmic Reticulum-Associated Degradation (ERAD) Has a Critical Role in Supporting Glucose-Stimulated Insulin Secretion in Pancreatic ß-Cells.

The molecular underpinnings of ß-cell dysfunction and death leading to diabetes are not fully elucidated. The objective of the current study was to investigate the role of endoplasmic reticulum-associated degradation (ERAD) in pancreatic ß-cells. Chemically induced ERAD deficiency in the rat insulinoma cell line INS-1 markedly reduced glucose-stimulated insulin secretion (GSIS). The mechanistic basis for this effect was studied in cells and mice lacking ERAD as a consequence of genetic ablation of the core ERAD protein SEL1L. Targeted disruption of SEL1L in INS-1 cells and in mouse pancreatic ß-cells impaired ERAD and led to blunted GSIS. Additionally, mice with SEL1L deletion in ß-cells were chronically hyperglycemic after birth and increasingly glucose intolerant over time. SEL1L absence caused an entrapment of proinsulin in the endoplasmic reticulum compartment in both INS-1 cells and mouse pancreatic ß-cells. Both folding-competent and folding-deficient proinsulin can physiologically interact with and be efficiently degraded by HRD1, the E3 ubiquitin ligase subunit of the ERAD complex. GSIS impairment in insulinoma cells was accompanied by a reduced intracellular Ca2+ ion level, overproduction of reactive oxygen species, and lowered mitochondrial membrane potential. Together, these findings suggest that ERAD plays a pivotal role in supporting pancreatic ß-cell function by targeting wild-type and folding-deficient proinsulin for proteosomal degradation. ERAD deficiency may contribute to the development of diabetes by affecting proinsulin processing in the ER, intracellular Ca2+ concentration, and mitochondrial function.

Hypothalamic ER-associated degradation regulates POMC maturation, feeding, and age-associated obesity.

Pro-opiomelanocortin (POMC) neurons function as key regulators of metabolism and physiology by releasing prohormone-derived neuropeptides with distinct biological activities. However, our understanding of early events in prohormone maturation in the ER remains incomplete. Highlighting the significance of this gap in knowledge, a single POMC cysteine-to-phenylalanine mutation at position 28 (POMC-C28F) is defective for ER processing and causes early onset obesity in a dominant-negative manner in humans through an unclear mechanism. Here, we report a pathologically important role of Sel1L-Hrd1, the protein complex of ER-associated degradation (ERAD), within POMC neurons. Mice with POMC neuron-specific Sel1L deficiency developed age-associated obesity due, at least in part, to the ER retention of POMC that led to hyperphagia. The Sel1L-Hrd1 complex targets a fraction of nascent POMC molecules for ubiquitination and proteasomal degradation, preventing accumulation of misfolded and aggregated POMC, thereby ensuring that another fraction of POMC can undergo normal posttranslational processing and trafficking for secretion. Moreover, we found that the disease-associated POMC-C28F mutant evades ERAD and becomes aggregated due to the presence of a highly reactive unpaired cysteine thiol at position 50. Thus, this study not only identifies ERAD as an important mechanism regulating POMC maturation within the ER, but also provides insights into the pathogenesis of monogenic obesity associated with defective prohormone folding.

Isolation and expression of zebrafish zinc-finger transcription factor gene tsh1.

We report the expression patterns of tsh1, a zebrafish homologue of the Drosophila homeotic gene teashirt. Expression of tsh1 is first detected at the 2-somite stage (10h post-fertilization, hpf) at the anterior end of the spinal cord. Expression expands toward the posterior spinal cord, and by the prim-5 stage (24 hpf) tsh1 transcripts are detected throughout spinal cord. Between the 14- and 25-somite stage (16-24 hpf), spinal cord expression shows a clear anterior boundary at the rostral margin of rhombomere 7. Around the prim-25 stage (36 hpf), while the spinal expression of tsh1 decreases, new expression is detected in the pectoral fin buds and dorsal forebrain. By the long-pec stage (48 hpf), spinal cord expression is undetectable, but strong expression is observed in the rhombencephalon, telencephalon, tectum opticum, midbrain-hindbrain boundary, in the first pharyngeal arch and in the eyes. This expression persists at least until the larval stages. Retinoic acid signaling influences tsh1 expression. Zebrafish tsh1 expression was induced in the anterior neural tube in embryos treated briefly with exogenous retinoic acid. Furthermore, tsh1 expression was down-regulated in the spinal cord in the zebrafish neckless mutant in which RA signaling is disrupted due to a missense mutation in the gene encoding retinaldehyde dehydrogenase type 2.

ER-associated degradation is required for vasopressin prohormone processing and systemic water homeostasis.

Peptide hormones are crucial regulators of many aspects of human physiology. Mutations that alter these signaling peptides are associated with physiological imbalances that underlie diseases. However, the conformational maturation of peptide hormone precursors (prohormones) in the ER remains largely unexplored. Here, we report that conformational maturation of proAVP, the precursor for the antidiuretic hormone arginine-vasopressin, within the ER requires the ER-associated degradation (ERAD) activity of the Sel1L-Hrd1 protein complex. Serum hyperosmolality induces expression of both ERAD components and proAVP in AVP-producing neurons. Mice with global or AVP neuron-specific ablation of Se1L-Hrd1 ERAD progressively developed polyuria and polydipsia, characteristics of diabetes insipidus. Mechanistically, we found that ERAD deficiency causes marked ER retention and aggregation of a large proportion of all proAVP protein. Further, we show that proAVP is an endogenous substrate of Sel1L-Hrd1 ERAD. The inability to clear misfolded proAVP with highly reactive cysteine thiols in the absence of Sel1L-Hrd1 ERAD causes proAVP to accumulate and participate in inappropriate intermolecular disulfide-bonded aggregates, promoted by the enzymatic activity of protein disulfide isomerase (PDI). This study highlights a pathway linking ERAD to prohormone conformational maturation in neuroendocrine cells, expanding the role of ERAD in providing a conducive ER environment for nascent proteins to reach proper conformation.

The ERV-9 LTR enhancer is not blocked by the HS5 insulator and synthesizes through the HS5 site non-coding, long RNAs that regulate LTR enhancer function.

A solitary long terminal repeat (LTR) of ERV-9 human endogenous retrovirus is located upstream of the HS5 site in the human beta-globin locus control region and possesses unique enhancer activity in erythroid K562 cells. In cells transfected with plasmid LTR-HS5-epsilonp-GFP, the LTR enhancer activates the GFP reporter gene and is not blocked by the interposed HS5 site, which has been reported to have insulator function. The LTR enhancer initiates synthesis of long RNAs from the LTR promoter through the intervening HS5 site into the epsilon-globin promoter and the GFP gene. Synthesis of the sense, long LTR RNAs is correlated with high level synthesis of GFP mRNA from the epsilon-globin promoter. Mutations of the LTR promoter and/or the epsilon-globin promoter show that (i) the LTR enhancer can autonomously initiate synthesis of LTR RNAs independent of the promoters and (ii) the LTR RNAs are not processed into GFP mRNA or translated into GFP. However, reversing the orientation of the LTR in plasmid (LTR)rev-HS5-epsilonp-GFP, thus reversing the direction of synthesis of LTR RNAs in the antisense direction away from the epsilon-globin promoter and GFP gene drastically reduces the level of GFP mRNA and thus LTR enhancer function. The results suggest that the LTR-assembled transcription machinery in synthesizing non-coding, LTR RNAs can reach the downstream epsilon-globin promoter to activate transcription of the GFP gene.

XBP1 Regulates the Biosynthetic Capacity of the Mammary Gland During Lactation by Controlling Epithelial Expansion and Endoplasmic Reticulum Formation.

Cells composing the mammary secretory compartment have evolved a high capacity to secrete not only proteins but also triglycerides and carbohydrates. This feature is illustrated by the mouse, which can secrete nearly twice its own weight in milk proteins, triglycerides and lactose over a short 20-day lactation. The coordination of synthesis and export of products in other secretory cells is orchestrated in part by the transcription factor X-box binding protein 1 (XBP1). To assess the role of XBP1 in mammary epithelial cells (MEC), we studied floxed XBP1 female mice lacking (wild type; WT) or expressing the Cre recombinase under the control of the ovine ß-lactoglobulin promoter (ΔXBP1(MEC)). Pregnant ΔXBP1(MEC) females had morphologically normal mammary development and gave birth to the same number of pups as WT mice. Their litters, however, suffered a weight gain deficit by lactation day 3 (L3)3 that grew to 80% by L14. ΔXBP1(MEC) dams had only modest changes in milk composition (-21% protein, +24% triglyceride) and in the expression of associated genes in isolated MEC. By L5, WT glands were fully occupied by dilated alveoli, whereas ΔXBP1(MEC) glands contained fewer, mostly unfilled alveoli and retained a prominent adipocyte population. The smaller epithelial compartment in ΔXBP1(MEC) glands was explained by lower MEC proliferation and increased apoptosis. Finally, endoplasmic reticulum ribbons were less abundant in ΔXBP1(MEC) at pregnancy day 18 and failed to increase in abundance by L5. Collectively, these results show that XBP1 is required for MEC population expansion during lactation and its ability to develop an elaborate endoplasmic reticulum compartment.