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Despite the prevalence of oncogenic Kras mutations in the earliest stages of pancreatic ductal adenocarcinoma, the cellular compartment in which oncogenic Kras initiates tumorigenesis remains unknown. To address this, we have gene targeted KrasG12D into the open reading frame of Mist1, a basic helix-loop-helix transcription factor that is expressed during pancreatic development and required for proper pancreatic acinar organization. Although the pancreata of Mist1KrasG12D/+ mutant mice predictably exhibited acinar metaplasia and dysplasia, the frequent death of these mice from invasive and metastatic pancreatic cancer with mixed histologic characteristics, including acinar, cystic, and ductal features, was unexpected and in contrast to previously described mutant mice that ectopically expressed the Kras oncogene in either acinar or ductal compartments. Interestingly, many of the mutant mice developed hepatocellular carcinoma, implicating Mist1KrasG12D/+ cells in both pancreatic and hepatic neoplasia. Concomitant Trp53+/− mutation cooperated with Mist1KrasG12D/+ to accelerate lethality and was associated with advanced histopathologic findings, including parenchymal liver metastasis. These findings suggest that Mist1-expressing cells represent a permissive compartment for transformation by oncogenic Kras in pancreatic tumorigenesis. (Cancer Res 2006; 66(1): 242-7)

Supplementary Figure 1 from Mist1-KrasG12D Knock-In Mice Develop Mixed Differentiation Metastatic Exocrine Pancreatic Carcinoma and Hepatocellular Carcinoma

Supplementary Legends from Mist1-KrasG12D Knock-In Mice Develop Mixed Differentiation Metastatic Exocrine Pancreatic Carcinoma and Hepatocellular Carcinoma

Supplementary Table 1 from Mist1-KrasG12D Knock-In Mice Develop Mixed Differentiation Metastatic Exocrine Pancreatic Carcinoma and Hepatocellular Carcinoma

One hundred years have passed since the discovery of insulin—an achievement that transformed diabetes from a fatal illness into a manageable chronic condition. The decades since that momentous achievement have brought ever more rapid innovation and advancement in diabetes research and clinical care. To celebrate the important work of the past century and help to chart a course for its continuation into the next, the Canadian Institutes of Health Research’s Institute of Nutrition, Metabolism and Diabetes and the U.S. National Institutes of Health’s National Institute of Diabetes and Digestive and Kidney Diseases recently held a joint international symposium, bringing together a cohort of researchers with diverse interests and backgrounds from both countries and beyond to discuss their collective quest to better understand the heterogeneity of diabetes and thus gain insights to inform new directions in diabetes treatment and prevention. This article summarizes the proceedings of that symposium, which spanned cutting-edge research into various aspects of islet biology, the heterogeneity of diabetic phenotypes, and the current state of and future prospects for precision medicine in diabetes.

Pancreatitis is a debilitating disease involving inflammation and fibrosis of the exocrine pancreas. Recurrent or chronic forms of pancreatitis are a significant risk factor for pancreatic ductal adenocarcinoma. While genetic factors have been identified for both pathologies, environmental stresses play a large role in their etiology. All cells have adapted mechanisms to handle acute environmental stress that alters energy demands. A common pathway involved in the stress response involves endoplasmic reticulum stress and the unfolded protein response (UPR). While rapidly activated by many external stressors, in the pancreas the UPR plays a fundamental biological role, likely due to the high protein demands in acinar cells. Despite this, increased UPR activity is observed in response to acute injury or following exposure to risk factors associated with pancreatitis and pancreatic cancer. Studies in animal and cell cultures models show the importance of affecting the UPR in the context of both diseases, and inhibitors have been developed for several specific mediators of the UPR. Given the importance of the UPR to normal acinar cell function, efforts to affect the UPR in the context of disease must be able to specifically target pathology vs. physiology. In this review, we highlight the importance of the UPR to normal and pathological conditions of the exocrine pancreas. We discuss recent studies suggesting the UPR may be involved in the initiation and progression of pancreatitis and PDAC, as well as contributing to chemoresistance that occurs in pancreatic cancer. Finally, we discuss the potential of targeting the UPR for treatment.

Background and AimsNon-alcoholic fatty liver disease and related hepatic syndromes affect up to one third of the adult population. The molecular mechanisms underlying NAFL etiology remain elusive. Nuclear Protein 1 (NUPR1) expression increases upon cell injury in all organs and recently we report its active participation in the activation of the Unfolded Protein Response (UPR). The UPR typically maintains protein homeostasis, but downstream mediators of the pathway regulate metabolic functions, including lipid metabolism. NUPR1 and UPR increase have been reported in obesity and liver pathologies and the goal of this study was to investigate the roles of NUPR1 in this context.MethodsWe used patient-derived liver biopsies and in vitro and in vivo NUPR1 loss of functions models. First, we analysed NUPR1 expression in a cohort of morbidly obese patients (MOPs), with either simple fatty liver (NAFL) or more severe steatohepatitis (NASH). Next, we explored the metabolic roles of NUPR1 in wild type (Nupr1+/+) or Nupr1 knockout mice (Nupr1-/-) fed ad libitum with a high fat diet (HFD) for up to 15 weeks.ResultsNUPR1 expression is inversely correlated to hepatic steatosis progression. We found that NUPR1 participates in the activation of PPAR-α signalling via UPR. PPAR-α signalling, is involved in the maintenance of fat metabolism and proper lipid homeostasis and energy balance. As PPAR-α signalling is controlled by UPR, collectively, these findings suggest a novel function for NUPR1 in protecting liver from metabolic distress by controlling lipid homeostasis, possibly through the UPR.ObjectiveNon-alcoholic fatty liver (NAFL) disease and related hepatic syndromes affect up to one third of the adult population in industrialised and developing countries. However, the molecular mechanisms underlying NAFL etiology remain elusive. Nuclear Protein 1 (NUPR1) expression increases upon cell injury in all organs including the liver. Recently, we report NUPR1 actively participates in activation of the Unfolded Protein Response (UPR). The UPR typically maintains protein homeostasis, but downstream mediators of the pathway regulate metabolic functions, including lipid metabolism. NUPR1 and UPR increase have been reported in obesity and liver pathologies and the goal of this study was to investigate the roles of NUPR1 in this context.DesignWe used patient-derived liver biopsies and in vitro and in vivo NUPR1 loss of functions models. First, we analysed NUPR1 expression in a cohort of morbidly obese patients (MOPs), with either simple fatty liver (NAFL) or more severe steatohepatitis (NASH). Next, we explored the metabolic roles of NUPR1 in wild type (Nupr1+/+) or Nupr1 knockout mice (Nupr1-/-) fed ad libitum with a high fat diet (HFD) for up to 15 weeks.ResultsNUPR1 expression is inversely correlated to hepatic steatosis progression. Mechanistically, we found NUPR1 participates in the activation of PPAR-α signalling via UPR. PPAR-α signalling, is involved in the maintenance of fat metabolism and proper lipid homeostasis and energy balance. As PPAR-α signalling is controlled by UPR, collectively, these findings suggest a novel function for NUPR1 in protecting liver from metabolic distress by controlling lipid homeostasis, possibly through the UPR.ConclusionsAs PPAR-α signalling is controlled by UPR, collectively, these findings suggest a novel function for NUPR1 in protecting liver from metabolic distress by controlling lipid homeostasis, possibly through the UPR.Lay summaryNUPR1 is activated during high caloric intake in both mice and patients. Decrease in expression or inhibition of NUPR1 worsens lipid deposition and hepatic damage.Graphical abstractHighlightsNUPR1 protects liver from high caloric intake hepatic damageThe function of NUPR1 in this context is to control the lipid homeostasis through the UPR and more specifically through PPAR-α signalling.NUPR1 could be used as a predictive marker for the gravity of NAFL progression. Moreover, as clinical interest is being raised around NUPR1 inhibitors to treat liver and pancreatic cancer, care should be taken in monitoring lipotoxic parameters.

International audience; Nuclear protein 1 (NUPR1) is a stress response protein overexpressed upon cell injury in virtually all organs including the exocrine pancreas. Despite NUPR1’s well-established role in the response to cell stress, the molecular and structural machineries triggered by NUPR1 activation remain largely debated. In this study, we uncover a new role for NUPR1, participating in the unfolded protein response (UPR) and the integrated stress response. Biochemical results and ultrastructural morphological observations revealed alterations in the UPR of acinar cells of germline-deleted NUPR1 murine models, consistent with the inability to restore general protein synthesis after stress induction. Bioinformatic analysis of NUPR1-interacting partners showed significant enrichment in translation initiation factors, including eukaryotic initiation factor (eIF) 2α. Co-immunoprecipitation and proximity ligation assays confirmed the interaction between NUPR1 and eIF2α and its phosphorylated form (p-eIF2α). Furthermore, our data suggest loss of NUPR1 in cells results in maintained eIF2α phosphorylation and evaluation of nascent proteins by click chemistry revealed that NUPR1-depleted PANC-1 cells displayed a slower poststress protein synthesis recovery when compared to wild-type. Combined, these data propose a novel role for NUPR1 in the integrated stress response pathway, at least partially through promoting efficient PERK branch activity and resolution through a unique interaction with eIF2α.

Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1

Pancreatic cancer remains among the deadliest forms of cancer with a 5 year survival rate less than 10%. With increasing numbers being observed, there is an urgent need to elucidate the pathogenesis of pancreatic cancer. While both contribute to disease progression, neither genetic nor environmental factors completely explain susceptibility or pathogenesis. Defining the links between genetic and environmental events represents an opportunity to understand the pathogenesis of pancreatic cancer. Epigenetics, the study of mitotically heritable changes in genome function without a change in nucleotide sequence, is an emerging field of research in pancreatic cancer. The main epigenetic mechanisms include DNA methylation, histone modifications and RNA interference, all of which are altered by changes to the environment. Epigenetic mechanisms are being investigated to clarify the underlying pathogenesis of pancreatic cancer including an increasing number of studies examining the role as possible diagnostic and prognostic biomarkers. These mechanisms also provide targets for promising new therapeutic approaches for this devastating malignancy.

The unfolded protein response (UPR) is activated in pancreatic pathologies and suggested as a target for therapeutic intervention. In this study, wxe examined Activating Transcription Factor 3 (ATF3), a mediator of the UPR which promotes acinar-to-ductal metaplasia (ADM) in response to pancreatic injury. Since ADM is an initial step in the progression to pancreatic ductal adenocarcinoma (PDAC), we hypothesized ATF3 is required for initiation and progression of PDAC. We generated mice carrying a germ line mutation ofAtf3(Atf3-/-) combined with acinar-specific induction of oncogenic KRAS (Ptf1acreERT/+KrasLSL-G12D).Atf3-/-mice with (termedAPK) and without KRASG12Dwere exposed to cerulein-induced pancreatitis. In response to recurrent pancreatitis,Atf3-/-mice showed decreased ADM and enhanced regeneration based on morphological and biochemical analysis. Similarly, an absence of ATF3 reduced spontaneous pancreatic intraepithelial neoplasia formation and PDAC inPtf1acreERT/+KrasLSL-G12Dmice. In response to injury, KRASG12Dbipassed the requirement for ATF3 with a dramatic loss in acinar tissue and PanIN formation observed regardless of ATF3 status. However, unlikePtf1acreERT/+KrasLSL-G12Dmice,APKmice exhibited a cachexia-like phenotype, did not progress through to PDAC, and showed altered pancreatic fibrosis and immune cell infiltration. These findings suggest a complex, multifaceted role for ATF3 in pancreatic cancer pathology.

NUPR1 is a stress response protein overexpressed upon cell injury in virtually all organs including the exocrine pancreas. Despite NUPR1’s well established role in the response to cell stress, the molecular and structural machineries triggered by NUPR1 activation remain largely unknown. In this study, we uncover an important role for NUPR1 in participating in the unfolded protein response pathway and the endoplasmic reticulum stress response. Biochemical results, confirmed by ultrastructural morphological observation, revealed alterations in the UPR in acinar cells of germline-deleted NUPR1 murine models, consistent with the inability to restore general protein translation. Bioinformatical analysis of NUPR1 interacting partners showed significant enrichment in translation initiation factors, including eukaryotic initiation factor (eIF) 2α. Co-immunoprecipitation and proximity ligation assays both confirmed interaction between NUPR1 and eIF2α and its phosphorylated form (p-eIF2α). Our. Moreover, our data also suggest loss of NUPR1 in cells results in maintained eIF2α phosphorylation and evaluation of nascent proteins by (peIF2α), and click chemistry revealed that NUPR1-depleted PANC-1 cells displayed a slower post stress protein translational recovery compared to wild-type. Combined, this data proposes a novel role for NUPR1 in the integrated stress response pathway, at least partially through promoting efficient PERK-branch activity and resolution through a unique interaction with eIF2α.SignificanceIn the pancreas, NUPR1 is required for a resolution of the ER stress response. During ER stress response, NUPR1 binds both eIF2α allowing for its dephosphorylation and restoration of new protein synthesis.HighlightsBiochemical analysis revealed a general reduction in the protein expression of downstream mediators of the unfolded protein response in the pancreas of mice lackingNupr1. This finding suggests a novel role for NUPR1 in the UPR/ER stress response.Ultrastructural analysis of pancreata revealed reduced morphological alterations in tunicamycin-treatedNupr1-/-mice compared toNupr1+/+mice consistent with a maintained block in general protein translation.Co-immunoprecipitation of tagged NUPR1 confirmed a novel interaction with eIF2α. Depletion of NUPR1 prolonged phosphorylation of eIF2α, suggesting it may be involved in attenuation of the PERK branch of the UPR.NUPR1-depleted PANC-1 cells displayed a slower recovery of protein translation following UPR activation

The unfolded protein response (UPR) is activated in pancreatic pathologies and suggested as a target for therapeutic intervention. In this study, we examined activating transcription factor 3 (ATF3), a mediator of the UPR that promotes acinar-to-ductal metaplasia (ADM) in response to pancreatic injury. Since ADM is an initial step in the progression to pancreatic ductal adenocarcinoma (PDAC), we hypothesized that ATF3 is required for initiation and progression of PDAC. We generated mice carrying a germline mutation of Atf3 (Atf3(−/−)) combined with acinar-specific induction of oncogenic KRAS (Ptf1a(creERT/+)Kras(G12D/+)). Atf3(−/−) mice with (termed APK) and without KRAS(G12D) were exposed to cerulein-induced pancreatitis. In response to recurrent pancreatitis, Atf3(−/−) mice showed decreased ADM and enhanced regeneration based on morphological and biochemical analysis. Similarly, an absence of ATF3 reduced spontaneous pancreatic intraepithelial neoplasia (PanIN) formation and PDAC in Ptf1a(creERT/+)Kras(G12D/+) mice. In response to injury, KRAS(G12D) bypassed the requirement for ATF3 with a dramatic loss in acinar tissue and PanIN formation observed regardless of ATF3 status. Compared to Ptf1a(creERT/+)Kras(G12D/+) mice, APK mice exhibited a significant decrease in pancreatic and total body weight, did not progress through to PDAC, and showed altered pancreatic fibrosis and immune cell infiltration. These findings suggest a complex, multifaceted role for ATF3 in pancreatic cancer pathology.

Organic anion-transporting polypeptides (OATPs) are membrane proteins that mediate cellular uptake of structurally diverse endogenous and exogenous compounds, including bile salts, thyroid and sex hormones, pharmacological agents, and toxins. Roles of OATPs in human liver are well established. Our recent report suggested the presence of the hepatic transporter OATP1B3 in human β cells. The aim of this study was to better characterize cellular localization and interindividual variation in OATP1B3 expression in human adult islets as a function of age, sex, and pancreatic disease, and to assess the expression of other OATPs. High transcript levels of OATP1B3, OATP2B1, OATP1A2, but not OATP1B1 were observed in isolated human adult islets. While OATP1B3 protein expression was variable, the carrier co-localized more frequently with glucagon-positive α cells than insulin-positive β cells in islets of normal pancreatic tissues from ten subjects using dual immunostaining. Moreover, OATP1B3 co-staining with endocrine cells was two- to three-fold higher in older (≥60 years) than younger (years) subjects. In comparison, in a subset of three individuals, OATP2B1 was primarily found in β cells, suggesting a distinct expression pattern for OATP1B3 and OATP2B1 in islets. Abundant OATP1B3 staining was also observed in islet as well as ductal cells of diseased tissues of patients with pancreatitis or pancreatic adenocarcinoma. Considering the abundance of key OATP carriers in β and α cells, potential implications of OATP transport in islet cell function may be suggested. Future studies are needed to gain insights into their specific endocrine roles as well as pharmacological relevance.

[Image: see text] The enzyme enhancer of zeste homologue 2 (EZH2) plays a catalytic role in histone methylation (H3K27me3), one of the epigenetic modifications that is dysregulated in cancer. The development of a positron emission tomography (PET) imaging agent targeting EZH2 has the potential to provide a method of stratifying patients for epigenetic therapies. In this study, we designed and synthesized a series of fluoroethyl analogs based upon the structure of EZH2 inhibitors UNC1999 and EPZ6438. Among the candidate compounds, 20b exhibited a high binding affinity to EZH2 (IC(50) = 6 nM) with selectivity versus EZH1 (IC(50) = 200 nM) by SAM competition assay, and furthermore, EZH2 inhibition was demonstrated in the pancreatic cancer cell line PANC-1 (IC(50) = 9.8 nM). [(18)F]20b was synthesized successfully and showed 5-fold higher uptake in PANC-1 cells than in MCF-7 cells. MicroPET imaging in a PANC-1 cell xenograft mouse model indicates that [(18)F]20b has specific binding to EZH2, which was identified by ex vivo Western blot analysis of the tumor tissue.

Proper regulation of cytosolic Ca(2+) is critical for pancreatic acinar cell function. Disruptions in normal Ca(2+) concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca(2+) homeostasis by promoting rapid Ca(2+) movement. Determining how expression of Ca(2+) modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas-specific isoform for Secretory Pathway Ca(2+) ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA-seq), chromatin immunoprecipitation for epigenetic modifications and promoter-reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last four exons of the Atp2c2 gene (Atp2c2c). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2's 24th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca(2+) . RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca(2+) levels. Combined, these results suggest SPCA2C affects Ca(2+) homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca(2+) ATPases. J. Cell. Physiol. 231: 2768-2778, 2016. © 2016 Wiley Periodicals, Inc.

Acinar cell exocytosis requires spatiotemporal Ca2+ signals regulated through endoplasmic reticulum (ER) stores, Ca2+ATPases, and store-operated Ca2+ entry (SOCE). The secretory pathway Ca2+ATPase 2 (SPCA2) interacts with Orai1, which is involved in SOCE and store independent Ca2+ entry (SICE). However, in the pancreas, only a C-terminally truncated form of SPCA2 (termed SPAC2C) exists. The goal of this study was to determine if SPCA2C effects Ca2+ homeostasis in a similar fashion to the full-length SPCA2. Using epitope-tagged SPCA2C (SPCA2CFLAG) expressed in HEK293A cells and Fura2 imaging, cytosolic [Ca2+] was examined during SICE, SOCE and secretagogue-stimulated signaling. Exogenous SPCA2C expression increased resting cytosolic [Ca2+], Ca2+ release in response to carbachol, ER Ca2+ stores, and store-mediated and independent Ca2+ influx. Co-IP detected Orai1-SPCA2C interaction, which was altered by co-expression of STIM1. Importantly, SPCA2C's effects on store-mediated Ca2+ entry were independent of Orai1. These findings indicate SPCA2C influences Ca2+ homeostasis through multiple mechanisms, some of which are independent of Orai1, suggesting novel and possibly cell-specific Ca2+ regulation.

International experts gathered at the Mayo Clinic (Rochester MN, USA) on February 27th-28th, 2017 for a meeting entitled 'Basic and Translational Facets of the Epigenetics of GI Diseases'. This workshop summarized recent advances on the role of epigenetics in the pathobiology of gastrointestinal (GI) diseases. Highlights of the meeting included recent advances on the involvement of different epigenetic mechanisms in malignant and nonmalignant GI disorders and the epigenetic heterogeneity exhibited in these diseases. The translational value of epigenetic drugs, as well as the current and future use of epigenetic changes (i.e., DNA methylation patterns) as biomarkers for early detection tools or disease stratification were also important topics of discussion.

© 2015, Springer-Verlag Berlin Heidelberg. Abstract: Osteoarthritis (OA) is a highly prevalent, disabling joint disease with no existing therapies to slow or halt its progression. Cartilage degeneration hallmarks OA pathogenesis, and pannexin 3 (Panx3), a member of a novel family of channel proteins, is upregulated during this process. The function of Panx3 remains poorly understood, but we consistently observed a strong increase in Panx3 immunostaining in OA lesions in both mice and humans. Here, we developed and characterized the first global and conditional Panx3 knockout mice to investigate the role of Panx3 in OA. Interestingly, global Panx3 deletion produced no overt phenotype and had no obvious effect on early skeletal development. Mice lacking Panx3 specifically in the cartilage and global Panx3 knockout mice were markedly resistant to the development of OA following destabilization of medial meniscus surgery. These data indicate a specific catabolic role of Panx3 in articular cartilage and identify Panx3 as a potential therapeutic target for OA. Lastly, while Panx1 has been linked to over a dozen human pathologies, this is the first in vivo evidence for a role of Panx3 in disease. Key message: Panx3 is localized to cartilage lesions in mice and humans.Global Panx3 deletion does not result in any developmental abnormalities.Mice lacking Panx3 are resistant to the development of osteoarthritis.Panx3 is a novel therapeutic target for the treatment of osteoarthritis.

A novel role for ATF3 is identified in acinar-to–duct cell metaplasia during pancreatic injury. ATF3 targets transcriptional regulators that affect acinar cell fate and reduces the severity of pancreatic injury. However, by doing so, ATF3 may also increase the susceptibility for progression to pancreatic adenocarcinoma., Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of >30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result, Atf3–/– pancreatic tissue displayed increased tissue damage and inflammatory cell infiltration at early time points during injury but, at later time points, showed reduced acinar-to–duct cell metaplasia. Thus our results reveal a critical role for ATF3 as a key regulator of the acinar cell transcriptional response during injury and may provide a link between chronic pancreatitis and PDAC.

Nonalcoholic fatty liver disease (NAFLD) alters drug response. We previously reported that NAFLD is associated with reduced in vivo CYP3A drug-metabolism activity and hepatic CYP3A4 expression in humans as well as mouse and human hepatoma models of the disease. Here, we investigated the role of the lipid- and glucose-modulating hormone fibroblast growth factor 21 (FGF21) in the molecular mechanism regulating CYP3A4 expression in NAFLD. In human subjects, mouse and cellular NAFLD models with lower CYP3A4 expression, circulating FGF21, or hepatic FGF21 mRNA levels were elevated. Administration of recombinant FGF21 or transient hepatic overexpression of FGF21 resulted in reduced liver CYP3A4 luciferase reporter activity in mice and decreased CYP3A4 mRNA expression and activity in cultured Huh7 hepatoma cells. Blocking canonical FGF21 signaling by pharmacological inhibition of MEK1 kinase in Huh7 cells caused de-repression of CYP3A4 mRNA expression with FGF21 treatment. Mice with high-fat diet-induced simple hepatic steatosis and lipid-loaded Huh7 cells had reduced nuclear localization of the pregnane X receptor (PXR), a key transcriptional regulator of CYP3A4 Furthermore, decreased nuclear PXR was observed in mouse liver and Huh7 cells after FGF21 treatment or FGF21 overexpression. Decreased PXR binding to the CYP3A4 proximal promoter was found in FGF21-treated Huh7 cells. An FGF21-PXR signaling pathway may be involved in decreased hepatic CYP3A4 metabolic activity in NAFLD.

Alteration in genetic expression is as important as gene mutation in cancer development and proliferation. Epigenetic changes affect gene expression without altering the DNA sequence. Histone deacetylase (HDAC), an enzyme facilitating histone remodelling, can lead to silencing of tumor suppressor genes making HDAC inhibitors viable anticancer drugs against tumors with increased activity of the enzyme. In this study we evaluated 18F-fluroacetamido-1-hexanoicanilide (18F-FAHA), an artificial HDAC substrate, as imaging probe of HDAC activity of human tumor xenografts in immunocompromised host mice. Human breast and melanoma cell lines, MDA-MB-468 and MDA-MB-435 respectively, known to overexpress HDAC activity were xenografted into immunocompromised mice and HDAC activity was imaged using 18F-FAHA. The melanoma group was treated with saline, SAHA (suberoylanilide hydroxamic acid, an approved anticancer HDAC inhibitor) in DMSO, or DMSO as positive control. Tracer kinetic modelling and SUV were used to estimate HDAC activity from dynamic PET data. Both breast tumor and melanoma group showed great variability in binding rate constant (BRC) of 18F-FAHA suggesting highly variable inter- and intra-tumoral HDAC activity. For the SAHA treated melanoma group, HDAC activity, as monitored by BRC of 18F-FAHA, decreased more than the two (positive and negative) control groups but not tumor growth. Our preliminary study showed that noninvasive PET imaging with 18F-FAHA has the potential to identify patients for whom treatment with HDAC inhibitors are appropriate, to assess the effectiveness of that treatment as an early marker of target reduction, and also eliminate the need for invasive tissue biopsy to individualize treatment.

Pancreatic ductal adenocarcinoma (PDAC) has a 5 year survival rate post-diagnosis of < 5%. Individuals with chronic pancreatitis (CP) are 20-fold more likely to develop PDAC, making it a significant risk factor for PDAC. While the relationship for the increased susceptibility to PDAC is unknown, loss of the acinar cell phenotype is common to both pathologies. Pancreatic acinar cells can dedifferentiate or trans-differentiate into a number of cell types including duct cells, β cells, hepatocytes and adipocytes. Knowledge of the molecular pathways that regulate this plasticity should provide insight into PDAC and CP. MIST1 (encoded by Bhlha15 in mice) is a transcription factor required for complete acinar cell maturation. The goal of this study was to examine the plasticity of acinar cells that do not express MIST1 (Mist1(-/-) ). The fate of acinar cells from C57Bl6 or congenic Mist1(-/-) mice expressing an acinar specific, tamoxifen-inducible Cre recombinase mated to Rosa26 reporter LacZ mice (Mist1(CreERT/-) R26r) was determined following culture in a three-dimensional collagen matrix. Mist1(CreERT/-) R26r acini showed increased acinar dedifferentiation, formation of ductal cysts and transient increases in PDX1 expression compared to wild-type acinar cells. Other progenitor cell markers, including Foxa1, Sox9, Sca1 and Hes1, were elevated only in Mist1(-/-) cultures. Analysis of protein kinase C (PKC) isoforms by western blot and immunofluorescence identified increased PKCe accumulation and nuclear localization of PKCδ that correlated with increased duct formation. Treatment with rottlerin, a PKCδ-specific inhibitor, but not the PKCe-specific antagonist eV1-2, reduced acinar dedifferentiation, progenitor gene expression and ductal cyst formation. Immunocytochemistry on CP or PDAC tissue samples showed reduced MIST1 expression combined with increased nuclear PKCδ accumulation. These results suggest that the loss of MIST1 is a common event during PDAC and CP and events that affect MIST1 function and expression may increase susceptibility to these pathologies.

Proper regulation of cytosolic Ca(2+) is critical for pancreatic acinar cell function. Disruptions in normal Ca(2+) concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca(2+) homeostasis by promoting rapid Ca(2+) movement. Determining how expression of Ca(2+) modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas-specific isoform for Secretory Pathway Ca(2+) ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA-seq), chromatin immunoprecipitation for epigenetic modifications and promoter-reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last four exons of the Atp2c2 gene (Atp2c2c). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2's 24th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca(2+) . RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca(2+) levels. Combined, these results suggest SPCA2C affects Ca(2+) homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca(2+) ATPases. J. Cell. Physiol. 231: 2768-2778, 2016. © 2016 Wiley Periodicals, Inc.

The seminal vesicle is a male accessory sex organ that develops from segments of the Wolffian duct adjacent to the urogenital sinus. It produces most of the seminal plasma in both humans and rodents. To date, very few transcription factors have been linked to the development and differentiation of seminal vesicles. In this study, we have examined the role of basic helix-loop-helix (BHLH) B8 transcription factor expressed at high levels in the adult seminal vesicle and during seminal gland differentiation. Immunofluorescent studies indicate that BHLHB8 is expressed within the epithelial layer of the seminal layer of the seminal vesicle following branching morphogenesis but prior to full maturation of cell morphology and function. Analysis of mice that do not express BHLHB8 (Bhlhb8−/−) indicates no deficiency in the initial development of the seminal vesicle. However, morphological and ultrastructural analysis indicates disruption of the epithelial cellular architecture. The seminal vesicle epithel...

Several animal models have been developed to investigate the pathobiology of pancreatitis, but few studies have examined the effects that altered pancreatic gene expression have in these models. In this study, the sensitivity to secretagogue-induced pancreatitis was examined in a mouse line that has an altered acinar cell environment due to the targeted deletion of Mist1. Mist1 is an exocrine specific transcription factor important for the complete differentiation and function of pancreatic acinar cells. Mice lacking the Mist1 gene [ Mist1 knockout (KO) mice] exhibit cellular disorganization and functional defects in the exocrine pancreas but no gross morphological defects. Following the induction of pancreatitis with caerulein, a CCK analog, we observed elevated serum amylase levels, necrosis, and tissue damage in Mist1 KO mice, indicating increased pancreatic damage. There was also a delay in the regeneration of acinar tissue in Mist1 KO animals. Molecular profiling revealed an altered activation of stress response genes in Mist1 KO pancreatic tissue compared with wild-type (WT) tissue following the induction of pancreatitis. In particular, Western blot analysis for activating transcription factor 3 and phosphorylated eukaryotic initiation factor 2α (eIF2α), mediators of endoplasmic reticulum (ER) stress, indicated limited activation of this pathway in Mist1 KO animals compared with WT controls. Conversely, Mist1 KO pancreatic tissue exhibits increased expression of growth arrest and DNA damage inducible 34 protein, an inhibitor of eIF2α phosphorylation, before and after the induction of pancreatitis. These finding suggest that activation of the ER stress pathway is a protective event in the progression of pancreatitis and highlight the Mist1 KO mouse line as an important new model for studying the molecular events that contribute to the sensitivity to pancreatic injury.

Cerebral edema in diabetic ketoacidosis (DKA-CE) occurs primarily in children and can develop during DKA therapy. The treatment factors contributing to DKA-CE remain elusive. Our objectives were to characterize an age-appropriate DKA mouse model and to determine which DKA therapies contribute to DKA-CE. Juvenile mice were briefly fed a high-fat diet and injected with two pancreatic beta-cell toxins: streptozocin and alloxan. Severe insulin and leptin deficiencies associated with hyperosmolar ketoacidosis rapidly developed, indicating DKA. DKA mice were treated with re-hydration +/- insulin and brain water content (BWC) measured as an indicator of DKA-CE. As expected, glucose and beta-OH-butyrate corrected in DKA mice that received rehydration and insulin. BWC significantly increased above control levels only in DKA mice that received combined insulin and bicarbonate therapy, indicating the development of DKA-CE. Microscopically, DKA-CE brains had perineuronal and perivascular edema, with microvacuolation in the white matter tracts. These results indicate that insulin-deficient juvenile mice develop biochemical changes that are similar to those of DKA in children. Increased BWC was observed only in DKA mice that received combined insulin and bicarbonate therapy, suggesting that rapid systemic alkalinization in the presence of insulin may contribute to DKA-CE.

The molecular mechanisms and metabolic pathways whereby the citrus flavonoid, naringenin, reduces dyslipidemia and improves glucose tolerance were investigated in C57BL6/J wild-type mice and fibroblast growth factor 21 (FGF21) null (Fgf21−/−) mice. FGF21 regulates energy homeostasis and the metabolic adaptation to fasting. One avenue of this regulation is through induction of peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc1a), a regulator of hepatic fatty acid oxidation and ketogenesis. Because naringenin is a potent activator of hepatic FA oxidation, we hypothesized that induction of FGF21 might be an integral part of naringenin's mechanism of action. Furthermore, we predicted that FGF21 deficiency would potentiate high-fat diet (HFD)-induced metabolic dysregulation and compromise metabolic protection by naringenin. The absence of FGF21 exacerbated the response to a HFD. Interestingly, naringenin supplementation to the HFD robustly prevented obesity in both genotypes. Gene expression analysis suggested that naringenin was not primarily targeting fatty acid metabolism in white adipose tissue. Naringenin corrected hepatic triglyceride concentrations and normalized hepatic expression of Pgc1a, Cpt1a, and Srebf1c in both wild-type and Fgf21−/− mice. HFD-fed Fgf21−/− mice displayed greater muscle triglyceride deposition, hyperinsulinemia, and impaired glucose tolerance as compared with wild-type mice, confirming the role of FGF21 in insulin sensitivity; however, naringenin supplementation improved these metabolic parameters in both genotypes. We conclude that FGF21 deficiency exacerbates HFD-induced obesity, hepatic steatosis, and insulin resistance. Furthermore, FGF21 is not required for naringenin to protect mice from HFD-induced metabolic dysregulation. Collectively these studies support the concept that naringenin has potent lipid-lowering effects and may act as an insulin sensitizer in vivo.

Acinar cells of the pancreas produce the majority of enzymes required for digestion and make up >90% of the cells within the pancreas. Due to a common developmental origin and the plastic nature of the acinar cell phenotype, these cells have been identified as a possible source of β cells as a therapeutic option for Type I diabetes. However, recent evidence indicates that acinar cells are the main source of pancreatic intraepithelial neoplasias (PanINs), the predecessor of pancreatic ductal adenocarcinoma (PDAC). The conversion of acinar cells to either β cells or precursors to PDAC is dependent on reprogramming of the cells to a more primitive, progenitor-like phenotype, which involves changes in transcription factor expression and activity, and changes in their epigenetic program. This review will focus on the mechanisms that promote acinar cell reprogramming, as well as the factors that may affect these mechanisms.

Thyroid hormone (TH) acts through specific receptors (TRs), which are conditional transcription factors, to induce fibroblast growth factor 21 (FGF21), a peptide hormone that is usually induced by fasting and that influences lipid and carbohydrate metabolism via local hepatic and systemic endocrine effects. While TH and FGF21 display overlapping actions when administered, including reductions in serum lipids, according to the current models these hormones act independentlyin vivo. In this study, we examined mechanisms of regulation of FGF21 expression by TH and tested the possibility that FGF21 is required for induction of hepatic TH-responsive genes. We confirm that active TH (triiodothyronine (T3)) and the TRβ-selective thyromimetic GC1 increase FGF21 transcript and peptide levels in mouse liver and that this effect requires TRβ. T3also induces FGF21 in cultured hepatocytes and this effect involves direct actions of TRβ1, which binds a TRE within intron 2 of FGF21. Gene expression profiles of WT andFgf21-knockout mice are very similar, indicating that FGF21 is dispensable for the majority of hepatic T3gene responses. A small subset of genes displays diminished T3response in the absence of FGF21. However, most of these are not obviously directly involved in T3-dependent hepatic metabolic processes. Consistent with these results, T3-dependent effects on serum cholesterol are maintained in theFgf21−/−background and we observe no effect of theFgf21-knockout background on serum triglycerides and glucose. Our findings indicate that T3regulates the genes involved in classical hepatic metabolic responses independently of FGF21.

Despite the prevalence of oncogenic Kras mutations in the earliest stages of pancreatic ductal adenocarcinoma, the cellular compartment in which oncogenic Kras initiates tumorigenesis remains unknown. To address this, we have gene targeted KrasG12D into the open reading frame of Mist1, a basic helix-loop-helix transcription factor that is expressed during pancreatic development and required for proper pancreatic acinar organization. Although the pancreata of Mist1KrasG12D/+ mutant mice predictably exhibited acinar metaplasia and dysplasia, the frequent death of these mice from invasive and metastatic pancreatic cancer with mixed histologic characteristics, including acinar, cystic, and ductal features, was unexpected and in contrast to previously described mutant mice that ectopically expressed the Kras oncogene in either acinar or ductal compartments. Interestingly, many of the mutant mice developed hepatocellular carcinoma, implicating Mist1KrasG12D/+ cells in both pancreatic and hepatic neoplasia. Concomitant Trp53+/− mutation cooperated with Mist1KrasG12D/+ to accelerate lethality and was associated with advanced histopathologic findings, including parenchymal liver metastasis. These findings suggest that Mist1-expressing cells represent a permissive compartment for transformation by oncogenic Kras in pancreatic tumorigenesis. (Cancer Res 2006; 66(1): 242-7)

Mist1 is an exocrine-specific transcription factor that is necessary for the establishment of cell organization and function of pancreatic acinar cells. While Mist1 is not expressed in the endocrine pancreas, the disorganized phenotype of the exocrine component may affect endocrine function. Therefore, we examined endocrine tissue morphology and function in Mist1-knockout (Mist1KO) mice. Endocrine function was evaluated using a glucose-tolerance test on 2–10-month-old female mice and revealed a significant reduction in glucose-clearing ability in 10-month-old Mist1KO mice compared with wild-type mice. Immunohistochemical analysis of islet hormone expression indicated that the decreased endocrine function was not due to a decrease in insulin-, glucagon- or somatostatin-expressing cells. However, a decrease in the size of islets in 10-month-old Mist1KO mice was observed along with a decrease in Glut-2 protein accumulation. These results suggest that the islets in Mist1KO mice are functionally compromised, likely accounting for the decreased glucose tolerance. Based on these findings, we have identified that the loss of a regulatory gene in the exocrine compartment can affect the endocrine component, providing a possible link between susceptibility for various pancreatic diseases.

Gap junctions are intercellular channels that provide direct passage of small molecules between adjacent cells. In pancreatic acini, the connexin26(Cx26) and connexin32 (Cx32) proteins form functional channels that coordinate the secretion of digestive enzymes. Although the function of Cx26/Cx32 gap junctions are well characterized, the regulatory circuits that control the spatial and temporal expression patterns of these connexin genes are not known. In an effort to identify the molecular pathways that regulate connexin gene expression, we examined Cx26 and Cx32 gene activities in mice lacking the basic helix-loop-helix transcription factor Mist1(Mist1KO). Mist1, Cx26 and Cx32 are co-expressed in most exocrine cell types, and acinar cells from Mist1KO mice exhibit a highly disorganized cellular architecture and an altered pattern of expression for several genes involved in regulated exocytosis. Analysis of Mist1KO mice revealed a dramatic decrease in both connexin proteins, albeit through different molecular mechanisms. Cx32 gene transcription was greatly reduced in all Mist1KO exocrine cells, while Cx26 gene expression remained unaffected. However, in the absence of Cx32 protein, Cx26 did not participate in gap junction formation, leading to a complete lack of intercellular communication among Mist1KO acinar cells. Additional studies testing Mist1 gene constructs in pancreatic exocrine cells confirmed that Mist1 transcriptionally regulates expression of the Cx32 gene. We conclude that Mist1 functions as a positive regulator of Cx32 gene expression and, in its absence, acinar cell gap junctions and intercellular communication pathways become disrupted.

The development of skeletal muscle is a highly regulated process governed by the four myogenic regulatory factors (MRFs) MyoD, myf-5, myogenin, and MRF4. While these factors exhibit some unique functions, part of their individual activity can be attributed to different temporal and spatial expression patterns. To delineate the factors that control expression of the MRFs, we have begun a molecular dissection of the MRF4 gene promoter. Through the generation of promoter/reporter gene constructs, we show that an 853bp fragment, residing 4kb upstream of the MRF4 transcriptional start site (853AV), is able to enhance expression of the basal MRF4 promoter 3-4-fold in myogenic cell cultures. Analysis of the 853AV enhancer in transgenic mice indicates that this region drives MRF4 gene expression primarily in fast muscle fibers, suggesting that the normal adult MRF4 expression pattern is regulated by a variety of control elements that may dictate fiber-type specificity.

Limb muscle development is characterized by the migration of muscle precursor cells from the somite followed by myoblast differentiation and the maturation of myotubes into distinct muscle fiber types. Previous in vitro experiments have suggested that rat limb myoblasts are composed of at least two distinct myoblast subpopulations that appear in the developing hindlimb at different developmental stages. These embryonic and fetal myoblast subpopulations are believed to generate primary and secondary myotubes, respectively. To test this hypothesis, cells obtained from embryonic day 14 (ED 14) and ED 20 rat hindlimbs were analyzed for myosin heavy chain expression after long-term differentiation in adult rat brains. Fetal myoblasts from ED 20 hindlimbs produced muscle fibers with a phenotype similar to that seen in tissue culture--predominantly fast myosin with a small proportion also coexpressing slow myosin. However, injection sites populated by embryonic myoblasts from ED 14 hindlimbs produced a different phenotype from that previously reported in culture, with fibers expressing an entire array of myosin isoforms. In addition, a subpopulation of fibers expressing exclusively slow myosin was found only in the embryonic injection sites. Our results support the existence of at least three myogenic subpopulations in early rat limb buds with only one exhibiting the capability to differentiate in vitro. These findings are consistent with a model of muscle fiber type development in which the fiber type potential of myoblast populations is established before differentiation into myotubes. This process establishes myogenic subpopulations that have restricted adaptive ranges regulated by both intrinsic and extrinsic factors.

Fibroblast growth factor 21 (FGF21) is a key regulator of metabolism under conditions of stress such as starvation, obesity, and hypothermia. Rapid induction of FGF21 is also observed in experimental models of pancreatitis, and FGF21 reduces tissue damage observed in these models, suggesting a nonmetabolic function. Pancreatitis is a debilitating disease with significant morbidity that greatly increases the risk of pancreatic ductal adenocarcinoma. The goals of this study were to examine the regulation and function of FGF21 in acinar cell injury, specifically in a mouse model of pancreatic injury ( Mist1 −/−). Mist1 −/− mice exhibit acinar cell disorganization, decreased acinar cell communication and exocytosis, and increased sensitivity to cerulein-induced pancreatitis (CIP). Examination of Fgf21 expression in Mist1 −/− mice by qRT-PCR, Northern blot, and Western blot analyses showed a marked decrease in pancreatic Fgf21 expression before and after induction of CIP compared with C57Bl/6 mice. To determine whether the loss of FGF21 accounted for the Mist1 −/− phenotypes, we generated Mist1 −/− mice overexpressing human FGF21 from the ApoE promoter ( Mist1 −/− ApoE-FGF21). Reexpression of FGF21 partially mitigated pancreatic damage in Mist1 −/− tissue based on reduced intrapancreatic enzyme activation, reduced expression of genes involved in fibrosis, and restored cell-cell junctions. Interestingly, alteration of Fgf21 expression in Mist1 −/− tissue was not simply due to a loss of direct transcriptional regulation by MIST1. Chromatin immunopreciptation indicated that the loss of Fgf21 in the Mist1 −/− pancreas is due, in part, to epigenetic silencing. Thus, our studies identify a new role for FGF21 in reducing acinar cell injury and uncover a novel mechanism for regulating Fgf21 gene expression.

Gene expression is affected by modifications to histone core proteins within chromatin. Changes in these modifications, or epigenetic reprogramming, can dictate cell fate and promote susceptibility to disease. The goal of this study was to determine the extent of epigenetic reprogramming in response to chronic stress that occurs following ablation of MIST1 (Mist1-/-), which is repressed in pancreatic disease. Chromatin immunoprecipitation for trimethylation of lysine residue 4 on histone 3 (H3K4Me3) in purified acinar cells from wild type and Mist1-/- mice was followed by Next Generation sequencing (ChIP-seq) or ChIP-qPCR. H3K4Me3-enriched genes were assessed for expression by qRT-PCR in pancreatic tissue before and after induction of cerulein-induced pancreatitis. While most of H3K4Me3-enrichment is restricted to transcriptional start sites, >25% of enrichment sites are found within, downstream or between annotated genes. Less than 10% of these sites were altered in Mist1-/- acini, with most changes in H3K4Me3 enrichment not reflecting altered gene expression. Ingenuity Pathway Analysis of genes differentially-enriched for H3K4Me3 revealed an association with pancreatitis and pancreatic ductal adenocarcinoma in Mist1-/- tissue. Most of these genes were not differentially expressed but several were readily induced by acute experimental pancreatitis, with significantly increased expression in Mist1-/- tissue relative to wild type mice. We suggest that the chronic cell stress observed in the absence of MIST1 results in epigenetic reprogramming of genes involved in promoting pancreatitis to a poised state, thereby increasing the sensitivity to events that promote disease. © 2014 Mehmood et al.

Mist1 is a basic helix-loop-helix transcription factor that represses E-box-mediated transcription. Previous studies have suggested that the Mist1 gene is expressed in a wide range of tissues, although a complete characterization of Mist1 protein accumulation in the adult organism has not been described. In an effort to identify specific cell types that contain the Mist1 protein, antibodies specific for Mist1 were generated and used in Western blot and immunohistochemical assays. Our studies show that the Mist1 protein is present in many different tissues but that it is restricted to cell types that are exclusively secretory in nature. Pancreatic acinar cells, serous or seromucous cells of the salivary glands, chief cells of the stomach, and secretory cells of the prostate and seminal vesicle show high levels of Mist1 protein, whereas nonserous exocrine cells, including the mucus-producing cells of the salivary glands, remain Mist1 negative. These results identify Mist1 as the first transcription factor that exhibits this unique serous-specific expression pattern and suggest that Mist1 may have a key role in establishing and maintaining a pathway responsible for the exocytosis of serous secretions.

To examine the relative importance of myoblast lineage and environmental influences on the development of muscle fiber types in vivo, the phenotype of muscle fibers formed from rat L6 myoblasts was examined following their injection into different regenerating adult muscles. Myoblasts were infected with a retroviral vector carrying a LacZ reporter gene and their fate in vivo was examined using a panel of antibodies against various myosin heavy chain (MyHC) isoforms. Since L6 myoblasts express IIX MyHC following differentiation in vitro, we wanted to determine if they would form IIX muscle fibers in vivo and whether innervation would alter this fate. Following injection, L6 cells either fused with each other to form homotypic fibers or fused with host muscle cells to form heterotypic fibers. Initially, homotypic fibers expressed embryonic MyHC-similar to L6 myotubes in vitro. However, by 4 weeks postinjection IIX MyHC had replaced embryonic MyHC as the predominant isoform. Single fiber analysis using an antibody specific for NCAM indicated that this transition was independent of innervation. Analysis of heterotypic fibers resulting from the incorporation of donor L6 myoblasts into host fast IIA and IIB fibers revealed that L6-derived nuclei express embryonic and IIX MyHCs for up to 8 weeks postinjection, often as nuclear domains surrounding L6 nuclei. These results suggest that MyHC expression in muscle fibers derived from L6 myoblasts is regulated, in part, by intrinsic factors that limit the fiber type potential of these cells in vivo.

The development of mammalian limb muscles involves the appearance and fusion of at least two separate populations of muscle precursor cells. These two populations, termed embryonic and fetal myoblasts, are first detected within the limb bud at different stages of development. We have previously demonstrated that, in the rat, each myoblast population expresses a unique pattern of myosin heavy chains (MyHCs) during differentiation in vitro (Pin and Merrifield [1993] Dev. Genet. 14:356-368). Embryonic myoblasts accumulate embryonic and slow MyHCs, whereas fetal myoblasts accumulate embryonic, neonatal, and adult fast MyHCs but not slow MyHC. To determine if the two populations can fuse with each other and whether the pattern of MyHC expression is altered in the resulting heterokaryons, embryonic and fetal myoblasts were labelled with the lipophilic dye PKH26, [3H]-thymidine, or 5-bromodeoxyuridine (BRDU) and cocultured for 24-48 hr. Our results demonstrate that fusion occurs between embryonic and fetal myoblasts in vitro. Moreover, analysis of the resulting heterokaryons revealed regionalized accumulations of MyHC around individual nuclei. Interestingly, these accumulations were typical of the default pattern of expression that individual nuclei would have normally expressed in single culture. Nuclei contributed by embryonic myoblasts were surrounded by localized accumulations of slow MyHC, whereas nuclei from fetal myoblasts were surrounded by neonatal/fast MyHC. The occurrence of such nuclear domains indicates that the myoblast-specific expression of MyHC isoforms is dictated by cis-acting factors established prior to fusion.

MRF4 is a muscle-specific tran- scription factor that belongs to a family of basic helix-loop-helix proteins known as the myogenic regulatory factors (MRFs). In vitro studies have shown that expression of the MRF4 gene is con- trolled by a proximal promoter element (2336 to 171) that binds the muscle-specific transcription factors MEF2 and myogenin to activate transcrip- tion. To examine further the regulatory elements necessary for endogenous MRF4 gene expression during development, transgenic mice were gener- ated that contained either a proximal MRF4 pro- moter-LacZ reporter gene (2336 MRF4-nLacZ )o r aMRF4-LacZ reporter gene containing 8.5 kb of 58 flanking sequence (28500 MRF4-nLacZ). Char- acterization of individual transgenic mouse lines throughout development revealed that expres- sion of both transgenes is restricted to skeletal muscle tissue. However, unlike previous in vitro data, the proximal promoter transgene exhibits only limited transcriptional activity at all devel- opmental time points, whereas the 28500 MRF4- nLacZ lines fully recapitulate the later develop- mental expression patterns and exhibit transcription in myotomal cells during somitic differentiation. Tissue culture analysis of myo- genic cells isolated from E12.5, E16.5, and adults confirmed that the 28500 MRF4-nLacZ transgene is expressed in greater than 90% of the myotubes for all myogenic populations. These results indi- cate that 8.5 kb of MRF4 58 flanking sequence contains all the regulatory elements necessary for lateMRF4expression and that at least some of these elements lie upstream of the 2336 proximal promoter. It is also likely that distant upstream regulatory sequences control early somiticMRF4 expression. These findings, coupled with previ- ous in vitro studies, suggest that the early and late developmental expression patterns of the MRF4 gene are controlled by distinct sets of regulatory elements. Dev. Dyn. 208:299-312, 1997.

Embryo cryopreservation has become a standard procedure in the practice of assisted reproduction. While routinely performed in IVF labs, the effects of embryo vitrification on the molecular mechanisms governing preimplantation development remain largely unknown. The endoplasmic reticulum stress (ER stress) response is an evolutionary conserved mechanism that cells employ to manage ER stress. ER stress can be defined as an imbalance between protein synthesis and secretion within the ER. The primary focus of this study was to investigate whether standard embryo manipulations, including embryo collection, culture and vitrification, result in activation of the ER stress pathway in vitro and to determine whether the embryo utilizes the unfolded protein response as an adaptive response. Our results indicate that the major ER stress pathway constituents are present at all stages of preimplantation development and that the activation of ER stress pathways can be induced at the 8-cell, morula and blastocyst stages. Additionally, we have demonstrated that the IRE1α arm of the ER Stress pathway is activated in freshly collected embryos but contrastingly, this ER Stress arm is not activated following embryo vitrification. It is important to understand the possible stresses that Assisted Reproductive Technologies place on the embryo and the mechanisms the embryo employs to adapt to these stresses. This study indicates that among the adaptive pathways available, cultured mammalian embryos can employ the ER stress pathway. Assisted reproduction techniques should be aware that their activities may induce the ER stress pathway in their patients' early embryos.