A TRPV 1‐to‐secretagogin regulatory axis controls pancreatic β‐cell survival by modulating protein turnover
2017; Springer Nature; Volume: 36; Issue: 14 Linguagem: Inglês
10.15252/embj.201695347
ISSN1460-2075
AutoresKatarzyna Maleńczyk, Fatima Girach, Edit Szodorai, Petter Storm, Åsa Segerstolpe, Giuseppe Tortoriello, R. Schnell, Jan Mulder, Roman A. Romanov, Erzsébet Borók, Fabiana Piscitelli, Vincenzo Di Marzo, Gábor Szabó, Rickard Sandberg, Stefan Kubicek, Gert Lübec, Tomas Hökfelt, Ludwig Wagner, Leif Groop, Tibor Harkany,
Tópico(s)Diet, Metabolism, and Disease
ResumoArticle21 June 2017free access A TRPV1-to-secretagogin regulatory axis controls pancreatic β-cell survival by modulating protein turnover Katarzyna Malenczyk Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Fatima Girach Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Edit Szodorai Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Petter Storm Department of Clinical Sciences, Diabetes and Endocrinology CRC, Skåne University Hospital Malmö, Malmö, Sweden Search for more papers by this author Åsa Segerstolpe Integrated Cardio Metabolic Centre, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Giuseppe Tortoriello Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Robert Schnell Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Jan Mulder Science for Life Laboratory, Karolinska Institutet, Solna, Sweden Search for more papers by this author Roman A Romanov Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Erzsébet Borók Department of Cognitive Neurobiology, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Fabiana Piscitelli Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Pozzuoli, Naples, Italy Search for more papers by this author Vincenzo Di Marzo Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Pozzuoli, Naples, Italy Search for more papers by this author Gábor Szabó Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary Search for more papers by this author Rickard Sandberg Integrated Cardio Metabolic Centre, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Stefan Kubicek CeMM Research Centre for Molecular Medicine, Vienna, Austria Search for more papers by this author Gert Lubec Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria Search for more papers by this author Tomas Hökfelt Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Ludwig Wagner University Clinic for Internal Medicine III, General Hospital Vienna, Vienna, Austria Search for more papers by this author Leif Groop Department of Clinical Sciences, Diabetes and Endocrinology CRC, Skåne University Hospital Malmö, Malmö, Sweden Search for more papers by this author Tibor Harkany Corresponding Author [email protected] orcid.org/0000-0002-6637-5900 Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Katarzyna Malenczyk Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Fatima Girach Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Edit Szodorai Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Petter Storm Department of Clinical Sciences, Diabetes and Endocrinology CRC, Skåne University Hospital Malmö, Malmö, Sweden Search for more papers by this author Åsa Segerstolpe Integrated Cardio Metabolic Centre, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Giuseppe Tortoriello Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Robert Schnell Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Jan Mulder Science for Life Laboratory, Karolinska Institutet, Solna, Sweden Search for more papers by this author Roman A Romanov Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Erzsébet Borók Department of Cognitive Neurobiology, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Fabiana Piscitelli Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Pozzuoli, Naples, Italy Search for more papers by this author Vincenzo Di Marzo Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Pozzuoli, Naples, Italy Search for more papers by this author Gábor Szabó Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary Search for more papers by this author Rickard Sandberg Integrated Cardio Metabolic Centre, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Stefan Kubicek CeMM Research Centre for Molecular Medicine, Vienna, Austria Search for more papers by this author Gert Lubec Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria Search for more papers by this author Tomas Hökfelt Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Ludwig Wagner University Clinic for Internal Medicine III, General Hospital Vienna, Vienna, Austria Search for more papers by this author Leif Groop Department of Clinical Sciences, Diabetes and Endocrinology CRC, Skåne University Hospital Malmö, Malmö, Sweden Search for more papers by this author Tibor Harkany Corresponding Author [email protected] orcid.org/0000-0002-6637-5900 Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Author Information Katarzyna Malenczyk1,2, Fatima Girach1, Edit Szodorai1, Petter Storm3, Åsa Segerstolpe4, Giuseppe Tortoriello2,†, Robert Schnell5, Jan Mulder6, Roman A Romanov1,2, Erzsébet Borók7, Fabiana Piscitelli8, Vincenzo Di Marzo8, Gábor Szabó9, Rickard Sandberg4, Stefan Kubicek10, Gert Lubec11,†, Tomas Hökfelt2, Ludwig Wagner12, Leif Groop3 and Tibor Harkany *,1,2 1Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria 2Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden 3Department of Clinical Sciences, Diabetes and Endocrinology CRC, Skåne University Hospital Malmö, Malmö, Sweden 4Integrated Cardio Metabolic Centre, Karolinska Institutet, Huddinge, Sweden 5Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden 6Science for Life Laboratory, Karolinska Institutet, Solna, Sweden 7Department of Cognitive Neurobiology, Center for Brain Research, Medical University of Vienna, Vienna, Austria 8Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Pozzuoli, Naples, Italy 9Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary 10CeMM Research Centre for Molecular Medicine, Vienna, Austria 11Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria 12University Clinic for Internal Medicine III, General Hospital Vienna, Vienna, Austria †Present address: Life Technologies, Glasgow, UK †Present address: Neuroproteomics Laboratory, Science Park, Ilkovicova 8, Bratislava, Slovakia *Corresponding author. Tel: +43 1 40160 34050; Fax: +43 1 40160 934053; E-mail: [email protected] EMBO J (2017)36:2107-2125https://doi.org/10.15252/embj.201695347 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Ca2+-sensor proteins are generally implicated in insulin release through SNARE interactions. Here, secretagogin, whose expression in human pancreatic islets correlates with their insulin content and the incidence of type 2 diabetes, is shown to orchestrate an unexpectedly distinct mechanism. Single-cell RNA-seq reveals retained expression of the TRP family members in β-cells from diabetic donors. Amongst these, pharmacological probing identifies Ca2+-permeable transient receptor potential vanilloid type 1 channels (TRPV1) as potent inducers of secretagogin expression through recruitment of Sp1 transcription factors. Accordingly, agonist stimulation of TRPV1s fails to rescue insulin release from pancreatic islets of glucose intolerant secretagogin knock-out(−/−) mice. However, instead of merely impinging on the SNARE machinery, reduced insulin availability in secretagogin−/− mice is due to β-cell loss, which is underpinned by the collapse of protein folding and deregulation of secretagogin-dependent USP9X deubiquitinase activity. Therefore, and considering the desensitization of TRPV1s in diabetic pancreata, a TRPV1-to-secretagogin regulatory axis seems critical to maintain the structural integrity and signal competence of β-cells. Synopsis TRPV1 activates Sp1-mediated secretagogin transcription in pancreatic β-cells to regulate β-cell survival, ER stress and glucose tolerance. Single-cell RNA-seq maps the expression of TRP family members to pancreatic β-cells. Ca2+ entry through TRP family channels regulates secretagogin transcription in β-cells via the Ca2+-dependent transcription factor Sp1. Secretagogin knock-out mice are insensitive to the pharmacological activation of TRPV1 receptors. Secretagogin knock-out mice are glucose intolerant and suffer from endoplasmic reticulum stress due to the breakdown of protein chaperone availability. In healthy and type 2 diabetic human β-cells secretagogin mRNA expression correlates with those of insulin, ATF4 and CHOP. Ca2+-bound secretagogin interacts with USP9X and USP7 to regulate β-cell turnover. Pharmacological modulation of protein degradation rescues β-cell viability. Introduction Life-long maintenance of the cellular integrity of Langerhans islets is critical for biphasic hormone release and therefore adequate responses to environmental and metabolic demands. β-cells produce insulin, and it is their progressive demise that is the causative basis of both type 1 and 2 diabetes (Deng et al, 2004; Ize-Ludlow et al, 2011). Despite intense research on restorative strategies to facilitate cell turnover or the expansion of the remnant β-cell pool, available solutions amenable to diabetic conditions remain limited. The lack of breakthroughs is particularly concerning when considering the manifold receptor-dependent signalling events modulating β-cell survival. Changes in intracellular Ca2+ levels regulate many cellular processes within pancreatic β-cells, ranging from transcription (German et al, 1990), protein folding (Creutz et al, 1994; Lievremont et al, 1997) and insulin exocytosis (Henquin et al, 2002) to the control of cell fate (Skrzypski et al, 2015). Translation of Ca2+ signals into physiologically meaningful output relies, in part, on Ca2+-sensor proteins, which upon Ca2+ binding undergo conformational changes and can phasically activate or inactivate their specific target partners via protein–protein interactions (Nelson & Chazin, 1998; Shen et al, 2008). To be available in sufficient amounts to decode Ca2+ signals, the intracellular turnover of Ca2+-sensors themselves may be Ca2+ regulated (van Der Luit et al, 1999), thus allowing precise control over their activity. Therefore, the coupling of receptor-mediated Ca2+ signalling to transcriptional programmes regulating Ca2+-sensor availability in β-cells represents an appealing yet modestly explored niche to integrate β-cell survival and functionality in type 2 diabetes. Secretagogin is an EF-hand Ca2+-sensor protein, which is amongst the most abundant proteins in pancreatic β-cells (Wagner et al, 2000). Primarily, secretagogin is thought to interact with cytoskeletal proteins (Maj et al, 2010; Yang et al, 2016), soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) components (e.g. SNAP-25, synaptobrevins; Rogstam et al, 2007; Romanov et al, 2015) and cargo proteins (Bauer et al, 2011) to modulate regulated insulin exocytosis. Accordingly, secretagogin loss-of-function studies in vitro show negative correlation with insulin release (Wagner et al, 2000; Hasegawa et al, 2013; Yang et al, 2016). Secondly, secretagogin has been implicated in the regulation of β-cell proliferation (Wagner et al, 2000). Therefore, and even if causality between secretagogin and insulin expression and release exists, the SNARE interactome of secretagogin alone is likely insufficient to chiefly account for the progressive loss of pancreatic β-cells in diabetes. Instead, the analysis of alternative regulatory pathways seems warranted, particularly to map receptor-to-gene transcription axes impacting the Ca2+-dependent regulation of cell turnover. Here, we combined bulk mRNA expression profiling of pancreatic islets obtained from a human cohort and single-cell RNA-seq from diabetic donors to establish an inverse correlation between secretagogin expression and the incidence of type 2 diabetes. To determine the identity of Ca2+-permeable channels regulating secretagogin function in β-cells, we focused on TRP family members that can fine-tune Ca2+-dependent transcriptional elements. Pharmacological probing of TRP channels revealed that transient receptor potential type 1 vanilloid channels (TRPV1) are particularly poised to induce secretagogin mRNA and protein expression. We then combined in silico models, secretagogin promoter assays, biochemistry and mouse genetics to establish that Sp1-dependent promoter activation is causal to TRPV1-mediated secretagogin expression. Next, we generated secretagogin−/− mice that by 6 weeks of age display glucose intolerance, which coincides with the progressive loss of their β-cell mass. By using global proteomics and histochemical tools, we find that Ca2+-bound secretagogin can coordinate protein folding and turnover. Accordingly, secretagogin−/− mice exhibit disrupted protein chaperonin activity and significant endoplasmic reticulum (ER) stress. Likewise, secretagogin mRNA levels in pancreatic islets from human diabetic donors negatively correlate with activating transcription factor 4 (ATF4) and CCAAT-enhancer-binding protein homologous protein (CHOP) expression. Cellular models and acute genetic manipulations reveal that secretagogin deletion renders its interacting partners, ubiquitin carboxyl-terminal hydrolases (USP9X and USP7) inactive, thus shifting the balance of β-cell proliferation/death towards increased apoptosis. Therefore, a ubiquitously Ca2+-driven TRPV1-to-secretagogin transcriptional axis is suggested as a signal transduction pathway whose deregulation can curtail the life-long structural integrity and functional competence of pancreatic β-cells. Results Secretagogin is a molecular predictor of type 2 diabetes Secretagogin is a Ca2+-sensor protein, and when silenced in vitro negatively couples to insulin secretion (Wagner et al, 2000; Hasegawa et al, 2013; Yang et al, 2016). Secretagogin mRNA and protein expression are preferentially associated with endocrine cell identities, including hormone or neuropeptide-producing cells in brain, pituitary, gastrointestinal tract and pancreas in both human and mouse (Fig EV1A and B; reviewed in: Alpar et al, 2012). More specifically, secretagogin immunoreactivity was seen in glucagon+/α-cells, insulin+/β-cells, somatostatin+/δ-cells and pancreatic polypeptide+/PP-cells in the islets of Langerhans in mouse (Fig EV1C). Thus, secretagogin is a ubiquitous cellular mark in the endocrine pancreas. Nevertheless, this study only focuses on dissecting secretagogin function in differentiated β-cells. Click here to expand this figure. Figure EV1. Body-wide secretagogin distribution in human and mouse Secretagogin expression in 12 human and mouse tissue types based on correlated RNA-seq and cap analysis of gene expression (CAGE) data. Data are expressed either as reads per kilobase of transcript per million mapped reads (RPKM) or tags per million (TPM). In human, secretagogin is mainly expressed in the CNS, endocrine glands (pancreas and pituitary) and the gastrointestinal tract. A similar expression pattern is observed in mouse with the exception of the cerebellum, which lacks secretagogin expression. Immunohistochemistry reveals secretagogin expression in a subset of (inter)neuron-like cells in the cerebral cortex and molecular layer of the cerebellum, endocrine pancreas, enteroendocrine cells of the stomach, small intestine and colon, as well as olfactory bulb of human subjects. Scale bars = 100 μm. Secretagogin was detected in glucagon+ α-cells, insulin+ β-cells, somatostatin (SST)+ δ-cells and some pancreatic polypeptide+ cells in adult mouse pancreas. Scale bars = 5 μm. Download figure Download PowerPoint Despite in vitro probing of its function in exocytosis earlier, the effects of secretagogin deficiency on glucose homoeostasis—particularly in humans—remain unknown. Therefore, we established whether secretagogin could partake in the development of a diabetic phenotype by analysing gene expression data from pancreatic islets of n = 32 subjects diagnosed with type 2 diabetes and n = 170 healthy controls (Fadista et al, 2014). We found a significant negative correlation between secretagogin mRNA expression and the incidence of diabetes (log2FC = −0.42, P = 0.0066; Fig 1A). Higher secretagogin mRNA expression also predicted lower glycated haemoglobin in blood (HbA1c; R2 = 0.059, P = 0.0097; Fig 1A1). Moreover, a significant positive correlation between secretagogin and insulin mRNA expression (R2 = 0.2, P = 2.7e−11; Fig 1A2) suggested a role for secretagogin in the hormonal control of glucose metabolism in vivo. Figure 1. TRP family channels induce secretagogin expression A–A2. Transcriptome analysis (bulk) of human (n = 202) pancreatic islets reveals decreased secretagogin (SCGN) expression in patients with type 2 diabetes (T2D; A). A negative correlation between secretagogin expression and the level of glycated haemoglobin (HbA1c) is shown (A1). Conversely, secretagogin mRNA levels positively correlate with insulin expression (A2). Data were presented as log2 counts per million (CPM). IGT, impaired glucose tolerance. Boxes represent 25th percentiles ± 90th percentiles, horizontal lines represent median values. B, B1. Single-cell RNA-seq analysis of TRP family members in β-cells from healthy (n = 6) and type 2 diabetic (n = 4) donors reveals retained TRP expression in β-cells from diabetic subjects. Data were expressed as log2 reads per kilobase of transcript per million mapped reads (RPKM). Boxes represent 25th percentiles ± 90th percentiles, horizontal lines represent mean values. C. Reverse-transcription PCR products of select TRP channels and Grin1 (encoding NMDA receptor subunit 1) in INS-1E cells. D. Acute agonist stimulation (30 min) of TRPM3 (CIM 0126; 1 μM), TRPV3 (2-APB; 25 μM) and TRPV1 (capsaicin; caps; 300 nM) promotes secretagogin expression in INS-1E cells with the most pronounced effect evoked by capsaicin. INS-1E cell depolarization with KCl (30 mM, 30 min) decreases secretagogin expression, which was reversed by NMDA. E. Capsazepine, a TRPV1 antagonist (10 μM), occluded capsaicin-induced secretagogin expression at both 30 and 120 min. F. Long-term (2–12 h) stimulation of INS-1E cells with capsaicin increases secretagogin protein content. Quantitative data reflect fold changes in SCGN signal intensity normalized to tubulin. G. Capsaicin fails to increase secretagogin mRNA expression in Ca2+-free media. Representative immunoblots are shown. Data information: Data were expressed as means ± s.d. from triplicate experiments (D–G). In (B, B1) **P < 0.01 calculated by Mann–Whitney rank-sum test. In (D–F) ***P < 0.001, **P < 0.01, *P < 0.05 calculated with pairwise comparisons/one-way ANOVA. Source data are available online for this figure. Source Data for Figure 1 [embj201695347-sup-0009-SDataFig1.pdf] Download figure Download PowerPoint TRP channels regulate secretagogin mRNA expression Ca2+-sensors are regulated by ionic conductances through Ca2+ permeable channels, thus allowing their efficient translation of environmental stimuli into adequately-tuned physiological cellular responses (Celio, 1990; Schwaller, 2010). Considering that ion binding of any sensor protein is not restricted to a single cation, we first tested secretagogin's metal ion specificity (the basis for being a “Ca2+-sensor”) using recombinant secretagogin (Fig EV2). We demonstrate seceretagogin's preference for bivalent members of the Beryllium group (Be, Ca, Mg, Sr) for protein-fold stabilization using the Thermofluor technique (Ericsson et al, 2006), which particularly favours Ca2+ over Mg2+ under quasi-physiological ion concentrations considering its ~100-fold higher affinity for Ca2+ (Rogstam et al, 2007). Click here to expand this figure. Figure EV2. Recombinant His6-tagged secretagogin only binds bivalent ions—calcium and its analogues A. Size exclusion chromatography confirming the purity of recombinant His6-tagged secretagogin. B. Protein folding integrity confirmed by circular dichroism spectroscopy returned the spectrum of a typical all-α fold as expected based on the known high-resolution structure of the zebrafish homologue (Bitto et al, 2009). C. Coomassie blue staining of purified His6-tagged secretagogin. D, E. Metal ion stabilization by secretagogin was investigated using the Thermofluor method (Ericsson et al, 2006). Typical thermal denaturation was observed in the presence of Ba2+, Ca2+, Mg2+, Sr2+ (all Ca2+-analogues; D), while the presence of other metal ions (E) resulted in curves where temperature-dependent unfolding could not be detected. All measurements were done in triplicate; representative results are shown. Download figure Download PowerPoint We hypothesized that Ca2+ entry could serve as an essential feedback for the expression of this Ca2+-sensor protein. Therefore, we searched for Ca2+ permeable channels that (i) can work independently or in tandem with glucose-regulated voltage-gated Ca2+ channels, which are lowly expressed in β-cells (Rorsman & Trube, 1986), (ii) their phasic activity can fine-tune β-cell Ca2+ signalling, (iii) their ligand levels are changing in type 2 diabetes and (iv) can affect cell proliferation (tissue size) during pancreas development or in pancreatic tumours since secretagogin is re-expressed upon tumorigenic transformation (Wagner et al, 2000; Birkenkamp-Demtroder et al, 2005; Lai et al, 2006). An appealing candidate satisfying the above criteria is the transient receptor potential (TRP) family of channels, which are evolutionarily conserved in the endocrine lineage, Ca2+ permeable, and their genetic ablation is implicated in tissue size control (Zhao et al, 2013; Riera et al, 2014; Malenczyk et al, 2015). When screening single-cell RNA-seq data in 270 β-cells from healthy and type 2 diabetic donors (Segerstolpe et al, 2016), we find that 17 TRP family members are expressed (> 0.001 RPKM; Fig 1B) with TRPC1 and TRPC4AP levels significantly increased in diabetes. Expression of most other channels was retained in β-cells from diabetic donors. We validated mRNA expression for Trpc1, Trpm3, Trpm5, Trpm7, Trpv1 and Trpv3 as representatives of the different TRP subfamilies (Clapham et al, 2001) as well as for Grin1 (Marquard et al, 2015) in INS-1E cells (Fig 1C). Next, we determined the effect of agonist stimulation of NMDA receptor, TRPM3, TRPV1 and TRPV3 with exogenous ligands (NMDA (20 μM), CIM 0126 (1 μM; Held et al, 2015), capsaicin (300 nM) and 2-APB (25 μM; Hu et al, 2009), respectively) on secretagogin mRNA expression. We applied 30 mM KCl to induce depolarization-dependent Ca2+ influx as control. After 30 min, CIM 0126 (P < 0.05), 2-APB (P < 0.01) and capsaicin (P < 0.001) increased secretagogin mRNA levels with capsaicin producing the most pronounced change (Fig 1D). In turn, KCl-induced passive Ca2+ overload reduced secretagogin expression, which was modulated by NMDA co-application (Fig 1D). These data show that temporally confined, receptor-mediated signalling events produce meaningful signals to induce secretagogin transcription. TRPV1-induced secretagogin accumulation in β-cells is Ca2+ dependent TRPV1s are particularly interesting since they are rapidly activated by anandamide, other endovanilloids and, for example, capsaicin (Di Marzo & De Petrocellis, 2012). In pancreas, both α- and β-cells express rate-limiting enzymes of anandamide metabolism and TRPV1s (Akiba et al, 2004; Malenczyk et al, 2013, 2015), which have been functionally implicated in the control of the intracellular Ca2+ level, insulin secretion and β-cell turnover (Akiba et al, 2004; De Petrocellis et al, 2007; Riera et al, 2014; Malenczyk et al, 2015). Anandamide is present in circulating blood, and its levels significantly increase in obesity and type 2 diabetes (Matias et al, 2006). In juvenile mouse plasma, we measured anandamide concentrations to be 20.4 ± 8.2 nM, superseding pKi for anandamide to activate TRPV1s (5.78 nM; Ross et al, 2001). We verified TRPV1 involvement in capsaicin-induced secretagogin mRNA expression (30 and 120 min time-points are shown; P < 0.05; Fig 1E) by demonstrating its sensitivity to capsazepine (TRPV1 antagonist, 10 μM) pre-treatment. We could also see the stimulatory effect of capsaicin on protein translation in INS-1E cells (2–12 h; Fig 1F). Finally, we confirmed the role of Ca2+ in the transcriptional regulation of secretagogin expression by showing that removal of extracellular Ca2+ precluded the TRPV1-dependent transcription of secretagogin (Fig 1G). Thus, a Ca2+-dependent mechanism might operate downstream from TRPV1 channels to control secretagogin function in β-cells. TRPV1s recruit Sp1 transcription factors to regulate secretagogin transcription We then analysed the mouse and human secretagogin promoter in silico to predict binding sites for transcription factors potentially involved in its expressional control (Tables EV1 and EV2). Since Ca2+ influx seems indispensable to increase secretagogin mRNA content in INS-1E cells, we focused on the Ca2+-dependent transcription factor Sp1 because its consensus binding sites were detected in the putative secretagogin promoter (−1,400 to +1) in both species (Fig 2A and A1). Notably, Sp1 activity has previously been linked to transmembrane channels permeable for bivalent cations, including TRPV1 (Moon et al, 2012). Figure 2. TRPV1 agonism promotes the Sp1-dependent activity of the predicted secretagogin promoter A, A1. In silico prediction of Sp1 transcription factor binding sites within the human and murine secretagogin promoters (up to −1,400 bps). (A1) Consensus recognition sequences exported from (A). B–B3. Capsaicin (caps; TRPV1 agonist; 300 nM for 30 min) induces Sp1 translocation to the nucleus (determined as increased Sp1 immunoreactivity) in INS-1E cells. Representative images are shown. Hoechst 33342 was used as nuclear counterstain. Scale bar = 5 μm. (B2) This capsaicin effect is blocked by capsazepine (cpz; TRPV1 antagonist, 10 μM). Capsaicin is also ineffective in the absence of extracellular Ca2+ (B3). Representative images for quantitative data are shown in Fig EV3. Data were expressed as means ± s.d. from triplicate experiments with n ≥ 100 cells/group quantified for (B2, B3). C, C1. Deletion of predicted Sp1 binding sites in the Scgn promoter (C) abrogates basal and capsaicin-induced (300 nM for 30 min) promoter activity defined as a ratio of firefly to Renilla luciferase chemiluminescence (3.5 h after stimulation; C1). Data were expressed as means ± s.d. from triplicate experiments. Data information: **P < 0.01, *P < 0.05; Student's t-test (C1) or one-way ANOVA (B2). Download figure Download PowerPoint Acute capsaicin stimulation (300 nM; 30 min) of TRPV1 receptors in INS-1E cells induced the nuclear translocation of Sp1. This response was capsazepine sensitive, confirming TRPV1 involvement (P < 0.05 vs. control; Figs 2B–B2 and EV3A–A3). Similarly, activation of TRPM3 and TRPV3 led to Sp1 translocation (Fig EV3C–C6) at magnitudes lesser than for TRPV1, corroborating our observations on secretagogin mRNA expression (Fig 1C). Likewise, TRPV1 stimulation under Ca2+-free conditions occluded Sp1 translocation (Figs 2B3 and EV3B–B3). Click here to expand this figure. Figure EV3. Activation of TRP channels promotes the nuclear translocation of Sp1 A–A3. Capsaicin (caps; TRPV1 agonist; 300 nM, 30 min) induces Sp1 translocation to the nuclei of INS-1E cells (determined as an increased level of
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