Critical Role of the Transcriptional Repressor Neuron-restrictive Silencer Factor in the Specific Control of Connexin36 in Insulin-producing Cell Lines

Abstract

Adjacent cells share ions, second messengers, small metabolites, and signaling molecules with a weight as high as 1000 Da (1) through intercellular channels that form gap junctions. This type of intercellular communication permits coordinated cellular activity and allows cells to review the functional state of their neighbors, a critical feature for the homeostasis of multicellular organisms (1-4). Intercellular channels result from the association of two half-channels named connexons, which are contributed to separately by each of two adjacent cells. Each connexon is an assembly of membrane proteins named connexins, which are encoded by a gene family consisting of at least 20 members (5, 6).It has been reported that Cx36 1 is the predominantly expressed connexin in rodent neuronal cells (7-9). Immunogold labeling using freeze-fracture replicas have shown that Cx36 was found only in neurons (10) and indicate that the specific expression of connexins by the different cell types of the nervous system may define separate pathways for neuronal versus glial gap junction communication (10 -12). We recently found that Cx36 is the predominant if not the sole connexin isoform expressed in insulin-producing ␤-cells of rat and mouse pancreatic islets (13,14). Recently, Cx36 content was modified in insulin-secreting cells by using an adenoviral gene transfer approach (15) or a lentivirus-mediated transduction of cx36 cDNA (16) in order to evaluate the contribution of Cx36 in the control of insulin secretion and content. These experiments have demonstrated that tight levels of Cx36 are crucial for proper function of insulin-producing cells. Moreover, stably transfected insulin-secreting MIN6 cells with a cx36 antisense cDNA impaired the synchronization of glucose-induced Ca( 2ϩ ) oscillations and insulin secretion in response to glucose (17). These data provided evidence that proper storage of insulin as well as the regulation of insulin release required that the levels of Cx36 be maintained within native values.The objective of the present study was to characterize the mechanisms of cell-specific expression of Cx36. The presence of several gene transcripts in pancreatic ␤-cells and neuronal cells was correlated with the absence in these cell types of a transcription factor named NRSF/REST (neuron-restrictive silencer factor/repressor element silencing transcription factor) (18). NRSF/REST binds to a 21-bp cis element named neuronal restrictive silencer element (NRSE). Through this NRSE, NRSF/REST has been identified as a negative regulatory system that silences neuronal gene expression in non-neuronal tissues, neuronal precursor cells, and certain differentiated

Functional significance of repressor element 1 silencing transcription factor (REST) target genes in pancreatic beta cells

“…Cells and islets were infected with adenoviruses as previously described [11], with a multiplicity of infection of 10 and 15, respectively. As judged by immunofluorescence and quantitative PCR detection of the REST transgene, this procedure typically resulted in the efficient transduction of 70 to 80% INS-1E cells and 30 to 40% primary islet cells.…”

“…Pelleted cells were lysed in an SDS buffer and submitted to sonication to obtain the desired chromatin length (~500 bp). Chromatin was precleared by addition of blocked protein A Sepharose (Amersham Bioscience Europe, Otelfingen, Switzerland) and the supernatants were immunoprecipitated overnight at 4°C with either polyclonal rabbit antibodies specific to human REST [11] or to irrelevant sterol regulatory element-binding protein-1 (SREBP-1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The protein-antibody complexes were collected by addition of protein A Sepharose for 1 h and then washed.…”

“…Transcripts levels were revealed using specific probes for rat Cx36 (also known as Gja9), β-actin [11], rat Munc18-1 (also known as Stxbp1), Syntaxin 1A and Rab3a. For the other genes, probes were obtained with cDNA from INS-1E cells or rat brain, which was amplified with specific primers (ESM Table 2).…”

“…Specific protein levels were revealed with polyclonal rabbit antibodies against: CX36 [11], synaptosomal-associated protein (SNAP) 25 (kindly provided by H. Hirling, EPFL, Lausanne, Switzerland) and SYT IV. Monoclonal antibodies against β-actin (Fluka Chemie) were used to normalise the signals.…”

“…However, it remains to be established how these genes participate in beta cell function. Investigating this molecular mechanism, we previously showed that in beta cells REST controls the expression of two neuron-specific genes that code for connexin 36 (CX36), a gap junction-forming protein participating in control of insulin secretion [11], and islet brain-1, a scaffold protein protecting beta cells against apoptosis via the c-Jun Nterminal kinase signalling pathway [12]. Since the whole set of REST target genes is involved in the control of insulin secretion in vitro [1], we have now examined the in vivo consequences of a gain of function of REST and have identified additional target genes that are significant to beta cell function.…”