Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameli... more Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or TGF-beta1 in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor beta (RARbeta), hepatocyte growth factor (HGF), cellular retinoic acid-binding protein II (CRABPII), vimentin, E-cadherin, Snail, and beta-catenin was assessed by immunoblotting. The cellular localization of vimentin and beta-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1458 genes after C-peptide exposure for 18 h or 48 h, respectively. From these, members of the antifibrotic retinoic acid (RA)- and HGF-signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARbeta, CRABPII, and HGF. We confirmed a role for RA in reversal of TGF-beta1-induced changes associated with epithelial-mesenchymal transition, including expression changes in Snail, E-cadherin, vimetin, and redistribution of beta-catenin. Importantly, these TGF-beta1-induced changes were inhibited by C-peptide. Further, effects of TGF-beta1 on Snail and E-cadherin expression were blocked by HGF, and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA-signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy.
Measurement of the intracellular Ca2+ concentration ([Ca2+]i) in fura-2-loaded single cells of th... more Measurement of the intracellular Ca2+ concentration ([Ca2+]i) in fura-2-loaded single cells of the human neuroblastoma line SH-SY5Y indicated coexpression of muscarinic and bradykinin receptors linked to activation of phosphoinositidase C (PIC). Both agonists elevated [Ca2+]i and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] levels in populations of adherent cells, although in cells used directly upon attainment of confluence the responses to carbachol were greater than those to bradykinin and displayed additional sustained components. This model system was used to examine heterologous interactions when a second PIC-linked agonist was added 100-300 sec after but in the continued presence of the first. Maximal (1 mM) carbachol concentrations abolished the elevation of [Ca2+]i produced by bradykinin but the muscarinic antagonist atropine (10 microM) restored the response, provided that extracellular Ca2+ was present throughout the experiment or was added before bradykinin. Carbachol also abolished bradykinin-mediated Ins(1,4,5)P3 elevation. In contrast, bradykinin did not influence [Ca2+]i or Ins(1,4,5)P3 responses to carbachol in the presence of extracellular Ca2+. In cells maintained at confluence for 2 weeks, the rapid peak elevations of [Ca2+]i and Ins(1,4,5)P3 levels induced by carbachol and bradykinin were approximately equivalent in magnitude. In these cells carbachol again abolished bradykinin-mediated elevation of [Ca2+]i but only attenuated, rather than abolished, the elevation of Ins(1,4,5)P3 levels. The [Ca2+]i and Ins(1,4,5)P3 responses to bradykinin were fully restored 100 sec after atropine only in the presence of extracellular Ca2+. Thus, depletion of an intracellular Ins(1,4,5)P3-sensitive Ca2+ store may underlie the ability of carbachol to produce not only heterologous desensitization of the [Ca2+]i elevation induced by bradykinin but also that of the Ins(1,4,5)P3 response. This suggests a feed-forward activation of PIC by Ca2+ released from Ins(1,4,5)P3-sensitive stores. Furthermore, studies in which Ins(1,4,5)P3-sensitive stores were depleted with thapsigargin and cells were challenged in the presence or absence of extracellular Ca2+ indicated that Ca2+, irrespective of its origin (intra- or extracellular), potentiated the Ins(1,4,5)P3 response to bradykinin alone. In cells maintained at confluence for 2 weeks, bradykinin was again unable to influence either [Ca2+]i or Ins(1,4,5)P3 responses to carbachol in the presence of Ca2+. This lack of heterologous desensitization may be due to the rapid, full, homologous desensitization of bradykinin receptors, compared with an incomplete homologous desensitization of muscarinic receptors.
A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G pro... more A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G protein pathways through a direct interaction with Gα subunits and stimulation of GTP hydrolysis. An RGS subfamily including RGS6, -7, -9, and -11, which contain a characteristic Gγ -like domain, also has the unique ability to interact with the G protein β subunit Gβ5. Here, we examined the behavior of Gβ5, RGS7, RGS9, and Gα in tissue extracts using immunoprecipitation and conventional chromatography. Native Gβ5 and RGS7 from brain, as well as photoreceptor-specific Gβ5L and RGS9, always co-purified as tightly associated dimers, and neither RGS-free Gβ5 nor Gβ5-free RGS could be detected. Co-expression in COS-7 cells of Gβ5 dramatically increased the protein level of RGS7 and vice versa, indicating that cells maintain Gβ5:RGS stoichiometry in a manner similar to Gβγ complexes. This mechanism is non-transcriptional and is based on increased protein stability upon dimerization. Thus, analysi...
This study examined cross talk between phospholipase C-coupled muscarinic M(3) and bradykinin B(2... more This study examined cross talk between phospholipase C-coupled muscarinic M(3) and bradykinin B(2) receptors coexpressed in Chinese hamster ovary (CHO) cells. Agonists of either receptor enhanced phosphoinositide signaling (which rapidly desensitized) and caused protein kinase C (PKC)-independent, homologous receptor phosphorylation. Muscarinic M(3) but not bradykinin B(2) receptors were also phosphorylated after phorbol ester activation of PKC. Consistent with this, muscarinic M(3) receptors were phosphorylated in a PKC-dependent fashion after bradykinin B(2) receptor activation, but muscarinic M(3) receptor activation did not influence bradykinin B(2) receptor phosphorylation. Despite heterologous phosphorylation of muscarinic M(3) receptors, phosphoinositide and Ca(2+) signaling were unaffected. In contrast, marked heterologous desensitization of bradykinin-mediated responses occurred despite no receptor phosphorylation. This desensitization was associated with a sustained compon...
The effects of protein kinase C (PKC) activation on muscarinic receptor-mediated phosphoinositide... more The effects of protein kinase C (PKC) activation on muscarinic receptor-mediated phosphoinositide and Ca2+ signalling were examined in the human neuroblastoma, SH-SY5Y. Carbachol evoked rapid transient elevations of Ins(1,4,5)P3 and intracellular [Ca2+] followed by lower sustained elevations. Phorbol 12,13-dibutyrate (PDBu) preferentially attenuated transient phases. Removal of the transplasmalemmal Ca2+ gradient coupled with depletion of intracellular Ca2+ stores with thapsigargin also reduced carbachol-mediated Ins(1,4,5)P3 accumulation. Under these conditions, PDBu virtually abolished Ins(1,4,5)P3 responses to carbachol thereby implicating both Ca(2+)- and PKC-sensitive components. PDBu also reduced agonist-mediated accumulation of inositol phosphates and depletion of lipids, thereby eliminating an effect of PKC on Ins(1,4,5)P3 metabolism or phosphoinositide synthesis. In electroporated cells, PDBu inhibited Ins(1,4,5)P3 accumulation mediated by carbachol or guanosine 5'-[gam...
Journal of Lipid Mediators and Cell Signalling, 1996
It is apparent that the phosphoinositide signalling pathway is subject to a variety of regulatory... more It is apparent that the phosphoinositide signalling pathway is subject to a variety of regulatory features which will ultimately dictate the magnitude and profile of cellular responses to agonist occupation of PIC-linked receptors. Our understanding of these mechanisms is far from complete but will be crucial in revealing both the specificity of receptor signalling and the integration of signals arising from the potentially wide variety of metabotropic and ionotropic receptors on individual cells.
Current concepts regarding the regulation and coupling of muscarinic m3 receptors to G-proteins a... more Current concepts regarding the regulation and coupling of muscarinic m3 receptors to G-proteins and various effecters are discussed. The last few years have provided much evidence that although muscarinic ml, m3 and m5 subtypes couple predominantly via pertussis toxin-insensitive G-proteins (Gq/l 1) to activate phosphoinositidase C (PIC), interactions with other G-proteins (Gi, Go, Gs) can be readily observed in cells expressing recombinant muscarinic receptors even at relatively low levels. The significance of this diversity and the potential for agonist "trafficking" could open up opportunities for novel approaches to selective agonist action.
Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient inges... more Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion and acts upon pancreatic b-cells potentiating glucose-stimulated insulin secretion and stimulating b-cell proliferation, differentiation, survival and gene transcription. These effects are mediated through the activation of multiple signal transduction pathways including the extracellular regulated kinase (ERK) pathway. We have previously reported that GLP-1 activates ERK through a mechanism dependent upon the influx of extracellular Ca 2+ through L-type voltage gated Ca 2+ channels (VGCC). However, the mechanism by which L-type VGCCs couple to the ERK signalling pathway in pancreatic b-cells is poorly understood. In this report, we characterise the relationship between L-type VGCC mediated changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ) and the activation of ERK, and demonstrate that the sustained activation of ERK (up to 30 min) in response to GLP-1 requires the continual activation of the L-type VGCC yet does not require a sustained increase in global [Ca 2+ ] i or Ca 2+ efflux from the endoplasmic reticulum. Moreover, sustained elevation of [Ca 2+ ] i induced by ionomycin is insufficient to stimulate the prolonged activation of ERK. Using the cell permeant Ca 2+ chelators, EGTA-AM and BAPTA-AM, to determine the spatial dynamics of L-type VGCC-dependent Ca 2+ signalling to ERK, we provide evidence that a sustained increase in Ca 2+ within the microdomain of the L-type VGCC is sufficient for signalling to ERK and that this plays an important role in GLP-1stimulated ERK activation. Citation: Selway J, Rigatti R, Storey N, Lu J, Willars GB, et al. (2012) Evidence That Ca 2+ within the Microdomain of the L-Type Voltage Gated Ca 2+ Channel Activates ERK in MIN6 Cells in Response to Glucagon-Like Peptide-1. PLoS ONE 7(3): e33004.
Recently we described a new, evolutionarily conserved cellular stress response characterized by a... more Recently we described a new, evolutionarily conserved cellular stress response characterized by a reversible reorganization of endoplasmic reticulum (ER) membranes that is distinct from canonical ER stress and the unfolded protein response (UPR). Apogossypol, a putative broad spectrum BCL-2 family antagonist, was the prototype compound used to induce this ER membrane reorganization. Following microarray analysis of cells treated with apogossypol, we used connectivity mapping to identify a wide range of structurally diverse chemicals from different pharmacological classes and established their ability to induce ER membrane reorganization. Such structural diversity suggests that the mechanisms initiating ER membrane reorganization are also diverse and a major objective of the present study was to identify potentially common features of these mechanisms. In order to explore this, we used hierarchical clustering of transcription profiles for a number of chemicals that induce membrane reorganization and discovered two distinct clusters. One cluster contained chemicals with known effects on Ca 2+ homeostasis. Support for this was provided by the findings that ER membrane reorganization was induced by agents that either deplete ER Ca 2+ (thapsigargin) or cause an alteration in cellular Ca 2+ handling (calmodulin antagonists). Furthermore, overexpression of the ER luminal Ca 2+ sensor, STIM1, also evoked ER membrane reorganization. Although perturbation of Ca 2+ homeostasis was clearly one mechanism by which some agents induced ER membrane reorganization, influx of extracellular Na + but not Ca 2+ was required for ER membrane reorganization induced by apogossypol and the related BCL-2 family antagonist, TW37, in both human and yeast cells. Not only is this novel, noncanonical ER stress response evolutionary conserved but so also are aspects of the mechanism of formation of ER membrane aggregates. Thus perturbation of ionic homeostasis is important in the regulation of ER membrane reorganization. Citation: Varadarajan S, Tanaka K, Smalley JL, Bampton ETW, Pellecchia M, et al. (2013) Endoplasmic Reticulum Membrane Reorganization Is Regulated by Ionic Homeostasis. PLoS ONE 8(2): e56603.
Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameli... more Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or TGF-beta1 in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor beta (RARbeta), hepatocyte growth factor (HGF), cellular retinoic acid-binding protein II (CRABPII), vimentin, E-cadherin, Snail, and beta-catenin was assessed by immunoblotting. The cellular localization of vimentin and beta-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1458 genes after C-peptide exposure for 18 h or 48 h, respectively. From these, members of the antifibrotic retinoic acid (RA)- and HGF-signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARbeta, CRABPII, and HGF. We confirmed a role for RA in reversal of TGF-beta1-induced changes associated with epithelial-mesenchymal transition, including expression changes in Snail, E-cadherin, vimetin, and redistribution of beta-catenin. Importantly, these TGF-beta1-induced changes were inhibited by C-peptide. Further, effects of TGF-beta1 on Snail and E-cadherin expression were blocked by HGF, and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA-signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy.
Journal of the American Society of Nephrology, 2006
Cell loss by apoptosis occurs in renal injury such as diabetic nephropathy. TNF-␣ is a cytokine t... more Cell loss by apoptosis occurs in renal injury such as diabetic nephropathy. TNF-␣ is a cytokine that induces apoptosis and has been implicated in the pathogenesis of diabetic nephropathy. The aim was to investigate whether C-peptide or insulin could modulate TNF-␣-mediated cell death in opossum kidney proximal tubular cells and to examine the mechanism(s) of any effects observed. C-peptide and insulin protect against TNF-␣-induced proximal tubular cell toxicity and apoptosis. Cell viability was analyzed by methylthiazoletetrazolium assay; cell viability was reduced to 60.8 ؎ 2.7% of control after stimulation with 300 ng/ml TNF-␣. Compromised cell viability was reversed by pretreatment with 5 nM C-peptide or 100 nM insulin. TNF-␣-induced apoptosis was detected by DNA nick-end labeling and by measuring histone associated DNA fragments using ELISA. By ELISA assay, 300 ng/ml TNF-␣ increased apoptosis by 145.8 ؎ 4.9% compared with controls, whereas 5 nM C-peptide and 100 nM insulin reduced apoptosis to 81.6 ؎ 4.8 and 77.4 ؎ 3.1% of control, respectively. The protective effects of C-peptide and insulin were associated with activation of NF-B. Activation of NF-B by C-peptide was pertussis toxin sensitive and dependent on activation of G␣ i . Phosphatidylinositol 3-kinase but not extracellular signal regulated mitogen-activated protein kinase mediated C-peptide and insulin activation of NF-B. The cytoprotective effects of both C-peptide and insulin were related to increased expression of TNF receptor-associated factor 2, the product of an NF-Bdependent survival gene. These data suggest that C-peptide and/or insulin activation of NF-B-regulated survival genes protects against TNF-␣-induced renal tubular injury in diabetes. The data further support the concept of C-peptide as a peptide hormone in its own right and suggest a potential therapeutic role for C-peptide.
Stimulation of gonadotropin-releasing hormone (GnRH) receptors with the GnRH analogue buserelin e... more Stimulation of gonadotropin-releasing hormone (GnRH) receptors with the GnRH analogue buserelin enhances expression of the zinc finger transcription factor Egr-1 in a pituitary gonadotroph cell line. The signaling cascade is blocked by overexpression of MAP kinase phosphatase-1 that dephosphorylates extracellular signal-regulated protein kinase in the nucleus. Chromatin immunoprecipitation experiments revealed that the phosphorylated form of Elk-1, a key regulator of gene transcription driven by serum response element (SRE), binds to the 5'-upstream region of the Egr-1 gene in buserelin-stimulated gonadotrophs. Expression of a dominant-negative mutant of Elk-1 completely blocked Egr-1 expression, indicating that Elk-1 connects the intracellular signaling cascade elicited by activation of GnRH receptors with transcription of the Egr-1 gene. GnRH receptor activation additionally induced the phosphorylation of CREB, which in its phosphorylated form bound to the Egr-1 gene. Expression of a dominant-negative mutant of CREB reduced GnRH receptor-induced upregulation of Egr-1 expression, indicating that CREB plays a role in the signaling pathway that regulates Egr-1 expression in gonadotrophs. We further identified the genes encoding basic fibroblast growth factor, tumor necrosis factor alpha, and transforming growth factor beta as bona fide target genes of Egr-1 in gonadotrophs. The analysis of gonadotroph cells that express--in addition to GnRH receptors--muscarinic M(3) acetylcholine receptors revealed that the nuclear events connecting GnRH receptors and muscarinic M(3) acetylcholine receptors with the Egr-1 gene are indistinguishable.
Peroxisome proliferator-activated receptor ␥ (PPAR␥) has key roles in the regulation of adipogene... more Peroxisome proliferator-activated receptor ␥ (PPAR␥) has key roles in the regulation of adipogenesis, inflammation, and lipid and glucose metabolism. C-peptide is believed to be inert and without appreciable biological functions. Recent studies suggest that C-peptide possesses multiple functions. The present study investigated the effects of insulin and C-peptide on PPAR␥ transcriptional activity in opossum kidney proximal tubular cells. Both insulin and C-peptide induced a concentration-dependent stimulation of PPAR␥ transcriptional activity. Both agents substantially augmented thiazolidinedione-stimulated PPAR␥ transcriptional activity. Neither insulin nor C-peptide had any effect on the expression levels of PPAR␥. GW9662, a PPAR␥ antagonist, blocked PPAR␥ activation by thiazolidinediones but had no effect on either insulin-or C-peptide-stimulated PPAR␥ transcriptional activity. Co-transfection of opossum kidney cells with dominant negative mitogen-activated protein kinase kinase significantly depressed basal PPAR␥ transcriptional activity but had no effect on that induced by either insulin or C-peptide. Both insulin-and C-peptide-stimulated PPAR␥ transcriptional activity were attenuated by wortmannin and by expression of a dominant negative phosphatidylinositol (PI) 3-kinase p85 regulatory subunit. In addition PI 3-kinase-dependent phosphorylation of PPAR␥ was observed after stimulation by C-peptide or insulin. C-peptide effects but not insulin on PPAR␥ transcriptional activity were abolished by pertussis toxin pretreatment. Finally both C-peptide and insulin positively control the expression of the PPAR␥-regulated CD36 scavenger receptor in human THP-1 monocytes. We concluded that insulin and C-peptide can stimulate PPAR␥ activity in a ligand-independent fashion and that this effect is mediated by PI 3-kinase. These results support a new and potentially important physiological role for C-peptide in regulation of PPAR␥-related cell functions.
We investigated the role played by agonist-mediated phosphorylation of the G(q/11)-coupled M(3)-m... more We investigated the role played by agonist-mediated phosphorylation of the G(q/11)-coupled M(3)-muscarinic receptor in the mechanism of activation of the mitogen-activated protein kinase pathway, ERK-1/2, in transfected Chinese hamster ovary cells. A mutant of the M(3)-muscarinic receptor, where residues Lys(370)-Ser(425) of the third intracellular loop had been deleted, showed a reduced ability to activate the ERK-1/2 pathway. This reduction was evident despite the fact that the receptor was able to couple efficiently to the phospholipase C second messenger pathway. Importantly, the ERK-1/2 responses to both the wild-type M(3)-muscarinic receptor and DeltaLys(370)-Ser(425) receptor mutant were dependent on the activity of protein kinase C. Our results, therefore, indicate the existence of two mechanistic components to the ERK-1/2 response, which appear to act in concert. First, the activation of protein kinase C through the diacylglycerol arm of the phospholipase C signaling pathway and a second component, absent in the DeltaLys(370)-Ser(425) receptor mutant, that is independent of the phospholipase C signaling pathway. The reduced ability of the DeltaLys(370)-Ser(425) receptor mutant to activate the ERK-1/2 pathway correlated with an approximately 80% decrease in the ability of the receptor to undergo agonist-mediated phosphorylation. Furthermore, we have previously shown that M(3)-muscarinic receptor phosphorylation can be inhibited by a dominant negative mutant of casein kinase 1alpha and by expression of a peptide corresponding to the third intracellular loop of the M(3)-muscarinic receptor. Expression of these inhibitors of receptor phosphorylation reduced the wild-type M(3)-muscarinic receptor ERK-1/2 response. We conclude that phosphorylation of the M(3)-muscarinic receptor on sites in the third intracellular loop by casein kinase 1alpha contributes to the mechanism of receptor activation of ERK-1/2 by working in concert with the diacylglycerol/PKC arm of the phospholipase C signaling pathway.
In this study we have quantitatively assessed the basal turnover of phosphatidylinositol 4,5-bisp... more In this study we have quantitatively assessed the basal turnover of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and M3-muscarinic receptor-mediated changes in phosphoinositides in the human neuroblastoma cell line, SH-SY5Y. We demonstrate that the polyphosphoinositides represent a minor fraction of the total cellular phosphoinositide pool and that in addition to rapid, sustained increases in [3H]inositol phosphates dependent upon the extent of receptor activation by carbachol, there are equally rapid and sustained reductions in the levels of polyphosphoinositides. Compared with phosphatidylinositol 4-phosphate (PtdIns(4)P), PtdIns(4,5)P2 was reduced with less potency by carbachol and recovered faster following agonist removal suggesting protection of PtdIns(4,5)P2 at the expense of PtdIns(4)P and indicating specific regulatory mechanism(s). This does not involve a pertussis toxin-sensitive G-protein regulation of PtdIns(4)P 5-kinase. Using wortmannin to inhibit PtdIns 4-kinase activity, we demonstrate that the immediate consequence of blocking the supply of PtdIns(4)P (and therefore PtdIns(4,5)P2) is a failure of agonist-mediated phosphoinositide and Ca2+ signaling. The use of wortmannin also indicated that PtdIns is not a substrate for receptor-activated phospholipase C and that 15% of the basal level of PtdIns(4,5)P2 is in an agonist-insensitive pool. We estimate that the agonist-sensitive pool of PtdIns(4,5)P2 turns over every 5 s (0.23 fmol/cell/min) during sustained receptor activation by a maximally effective concentration of carbachol. Immediately following agonist addition, PtdIns(4,5)P2 is consumed >3 times faster (0.76 fmol/cell/min) than during sustained receptor activation which represents, therefore, utilization by a partially desensitized receptor. These data indicate that resynthesis of PtdIns(4,5)P2 is required to allow full early and sustained phases of receptor signaling. Despite the critical dependence of phosphoinositide and Ca2+ signaling on PtdIns(4,5)P2 resynthesis, we find no evidence that this rate resynthesis is limiting for agonist-mediated responses.
Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameli... more Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or TGF-beta1 in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor beta (RARbeta), hepatocyte growth factor (HGF), cellular retinoic acid-binding protein II (CRABPII), vimentin, E-cadherin, Snail, and beta-catenin was assessed by immunoblotting. The cellular localization of vimentin and beta-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1458 genes after C-peptide exposure for 18 h or 48 h, respectively. From these, members of the antifibrotic retinoic acid (RA)- and HGF-signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARbeta, CRABPII, and HGF. We confirmed a role for RA in reversal of TGF-beta1-induced changes associated with epithelial-mesenchymal transition, including expression changes in Snail, E-cadherin, vimetin, and redistribution of beta-catenin. Importantly, these TGF-beta1-induced changes were inhibited by C-peptide. Further, effects of TGF-beta1 on Snail and E-cadherin expression were blocked by HGF, and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA-signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy.
Measurement of the intracellular Ca2+ concentration ([Ca2+]i) in fura-2-loaded single cells of th... more Measurement of the intracellular Ca2+ concentration ([Ca2+]i) in fura-2-loaded single cells of the human neuroblastoma line SH-SY5Y indicated coexpression of muscarinic and bradykinin receptors linked to activation of phosphoinositidase C (PIC). Both agonists elevated [Ca2+]i and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] levels in populations of adherent cells, although in cells used directly upon attainment of confluence the responses to carbachol were greater than those to bradykinin and displayed additional sustained components. This model system was used to examine heterologous interactions when a second PIC-linked agonist was added 100-300 sec after but in the continued presence of the first. Maximal (1 mM) carbachol concentrations abolished the elevation of [Ca2+]i produced by bradykinin but the muscarinic antagonist atropine (10 microM) restored the response, provided that extracellular Ca2+ was present throughout the experiment or was added before bradykinin. Carbachol also abolished bradykinin-mediated Ins(1,4,5)P3 elevation. In contrast, bradykinin did not influence [Ca2+]i or Ins(1,4,5)P3 responses to carbachol in the presence of extracellular Ca2+. In cells maintained at confluence for 2 weeks, the rapid peak elevations of [Ca2+]i and Ins(1,4,5)P3 levels induced by carbachol and bradykinin were approximately equivalent in magnitude. In these cells carbachol again abolished bradykinin-mediated elevation of [Ca2+]i but only attenuated, rather than abolished, the elevation of Ins(1,4,5)P3 levels. The [Ca2+]i and Ins(1,4,5)P3 responses to bradykinin were fully restored 100 sec after atropine only in the presence of extracellular Ca2+. Thus, depletion of an intracellular Ins(1,4,5)P3-sensitive Ca2+ store may underlie the ability of carbachol to produce not only heterologous desensitization of the [Ca2+]i elevation induced by bradykinin but also that of the Ins(1,4,5)P3 response. This suggests a feed-forward activation of PIC by Ca2+ released from Ins(1,4,5)P3-sensitive stores. Furthermore, studies in which Ins(1,4,5)P3-sensitive stores were depleted with thapsigargin and cells were challenged in the presence or absence of extracellular Ca2+ indicated that Ca2+, irrespective of its origin (intra- or extracellular), potentiated the Ins(1,4,5)P3 response to bradykinin alone. In cells maintained at confluence for 2 weeks, bradykinin was again unable to influence either [Ca2+]i or Ins(1,4,5)P3 responses to carbachol in the presence of Ca2+. This lack of heterologous desensitization may be due to the rapid, full, homologous desensitization of bradykinin receptors, compared with an incomplete homologous desensitization of muscarinic receptors.
A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G pro... more A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G protein pathways through a direct interaction with Gα subunits and stimulation of GTP hydrolysis. An RGS subfamily including RGS6, -7, -9, and -11, which contain a characteristic Gγ -like domain, also has the unique ability to interact with the G protein β subunit Gβ5. Here, we examined the behavior of Gβ5, RGS7, RGS9, and Gα in tissue extracts using immunoprecipitation and conventional chromatography. Native Gβ5 and RGS7 from brain, as well as photoreceptor-specific Gβ5L and RGS9, always co-purified as tightly associated dimers, and neither RGS-free Gβ5 nor Gβ5-free RGS could be detected. Co-expression in COS-7 cells of Gβ5 dramatically increased the protein level of RGS7 and vice versa, indicating that cells maintain Gβ5:RGS stoichiometry in a manner similar to Gβγ complexes. This mechanism is non-transcriptional and is based on increased protein stability upon dimerization. Thus, analysi...
This study examined cross talk between phospholipase C-coupled muscarinic M(3) and bradykinin B(2... more This study examined cross talk between phospholipase C-coupled muscarinic M(3) and bradykinin B(2) receptors coexpressed in Chinese hamster ovary (CHO) cells. Agonists of either receptor enhanced phosphoinositide signaling (which rapidly desensitized) and caused protein kinase C (PKC)-independent, homologous receptor phosphorylation. Muscarinic M(3) but not bradykinin B(2) receptors were also phosphorylated after phorbol ester activation of PKC. Consistent with this, muscarinic M(3) receptors were phosphorylated in a PKC-dependent fashion after bradykinin B(2) receptor activation, but muscarinic M(3) receptor activation did not influence bradykinin B(2) receptor phosphorylation. Despite heterologous phosphorylation of muscarinic M(3) receptors, phosphoinositide and Ca(2+) signaling were unaffected. In contrast, marked heterologous desensitization of bradykinin-mediated responses occurred despite no receptor phosphorylation. This desensitization was associated with a sustained compon...
The effects of protein kinase C (PKC) activation on muscarinic receptor-mediated phosphoinositide... more The effects of protein kinase C (PKC) activation on muscarinic receptor-mediated phosphoinositide and Ca2+ signalling were examined in the human neuroblastoma, SH-SY5Y. Carbachol evoked rapid transient elevations of Ins(1,4,5)P3 and intracellular [Ca2+] followed by lower sustained elevations. Phorbol 12,13-dibutyrate (PDBu) preferentially attenuated transient phases. Removal of the transplasmalemmal Ca2+ gradient coupled with depletion of intracellular Ca2+ stores with thapsigargin also reduced carbachol-mediated Ins(1,4,5)P3 accumulation. Under these conditions, PDBu virtually abolished Ins(1,4,5)P3 responses to carbachol thereby implicating both Ca(2+)- and PKC-sensitive components. PDBu also reduced agonist-mediated accumulation of inositol phosphates and depletion of lipids, thereby eliminating an effect of PKC on Ins(1,4,5)P3 metabolism or phosphoinositide synthesis. In electroporated cells, PDBu inhibited Ins(1,4,5)P3 accumulation mediated by carbachol or guanosine 5'-[gam...
Journal of Lipid Mediators and Cell Signalling, 1996
It is apparent that the phosphoinositide signalling pathway is subject to a variety of regulatory... more It is apparent that the phosphoinositide signalling pathway is subject to a variety of regulatory features which will ultimately dictate the magnitude and profile of cellular responses to agonist occupation of PIC-linked receptors. Our understanding of these mechanisms is far from complete but will be crucial in revealing both the specificity of receptor signalling and the integration of signals arising from the potentially wide variety of metabotropic and ionotropic receptors on individual cells.
Current concepts regarding the regulation and coupling of muscarinic m3 receptors to G-proteins a... more Current concepts regarding the regulation and coupling of muscarinic m3 receptors to G-proteins and various effecters are discussed. The last few years have provided much evidence that although muscarinic ml, m3 and m5 subtypes couple predominantly via pertussis toxin-insensitive G-proteins (Gq/l 1) to activate phosphoinositidase C (PIC), interactions with other G-proteins (Gi, Go, Gs) can be readily observed in cells expressing recombinant muscarinic receptors even at relatively low levels. The significance of this diversity and the potential for agonist "trafficking" could open up opportunities for novel approaches to selective agonist action.
Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient inges... more Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion and acts upon pancreatic b-cells potentiating glucose-stimulated insulin secretion and stimulating b-cell proliferation, differentiation, survival and gene transcription. These effects are mediated through the activation of multiple signal transduction pathways including the extracellular regulated kinase (ERK) pathway. We have previously reported that GLP-1 activates ERK through a mechanism dependent upon the influx of extracellular Ca 2+ through L-type voltage gated Ca 2+ channels (VGCC). However, the mechanism by which L-type VGCCs couple to the ERK signalling pathway in pancreatic b-cells is poorly understood. In this report, we characterise the relationship between L-type VGCC mediated changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ) and the activation of ERK, and demonstrate that the sustained activation of ERK (up to 30 min) in response to GLP-1 requires the continual activation of the L-type VGCC yet does not require a sustained increase in global [Ca 2+ ] i or Ca 2+ efflux from the endoplasmic reticulum. Moreover, sustained elevation of [Ca 2+ ] i induced by ionomycin is insufficient to stimulate the prolonged activation of ERK. Using the cell permeant Ca 2+ chelators, EGTA-AM and BAPTA-AM, to determine the spatial dynamics of L-type VGCC-dependent Ca 2+ signalling to ERK, we provide evidence that a sustained increase in Ca 2+ within the microdomain of the L-type VGCC is sufficient for signalling to ERK and that this plays an important role in GLP-1stimulated ERK activation. Citation: Selway J, Rigatti R, Storey N, Lu J, Willars GB, et al. (2012) Evidence That Ca 2+ within the Microdomain of the L-Type Voltage Gated Ca 2+ Channel Activates ERK in MIN6 Cells in Response to Glucagon-Like Peptide-1. PLoS ONE 7(3): e33004.
Recently we described a new, evolutionarily conserved cellular stress response characterized by a... more Recently we described a new, evolutionarily conserved cellular stress response characterized by a reversible reorganization of endoplasmic reticulum (ER) membranes that is distinct from canonical ER stress and the unfolded protein response (UPR). Apogossypol, a putative broad spectrum BCL-2 family antagonist, was the prototype compound used to induce this ER membrane reorganization. Following microarray analysis of cells treated with apogossypol, we used connectivity mapping to identify a wide range of structurally diverse chemicals from different pharmacological classes and established their ability to induce ER membrane reorganization. Such structural diversity suggests that the mechanisms initiating ER membrane reorganization are also diverse and a major objective of the present study was to identify potentially common features of these mechanisms. In order to explore this, we used hierarchical clustering of transcription profiles for a number of chemicals that induce membrane reorganization and discovered two distinct clusters. One cluster contained chemicals with known effects on Ca 2+ homeostasis. Support for this was provided by the findings that ER membrane reorganization was induced by agents that either deplete ER Ca 2+ (thapsigargin) or cause an alteration in cellular Ca 2+ handling (calmodulin antagonists). Furthermore, overexpression of the ER luminal Ca 2+ sensor, STIM1, also evoked ER membrane reorganization. Although perturbation of Ca 2+ homeostasis was clearly one mechanism by which some agents induced ER membrane reorganization, influx of extracellular Na + but not Ca 2+ was required for ER membrane reorganization induced by apogossypol and the related BCL-2 family antagonist, TW37, in both human and yeast cells. Not only is this novel, noncanonical ER stress response evolutionary conserved but so also are aspects of the mechanism of formation of ER membrane aggregates. Thus perturbation of ionic homeostasis is important in the regulation of ER membrane reorganization. Citation: Varadarajan S, Tanaka K, Smalley JL, Bampton ETW, Pellecchia M, et al. (2013) Endoplasmic Reticulum Membrane Reorganization Is Regulated by Ionic Homeostasis. PLoS ONE 8(2): e56603.
Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameli... more Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or TGF-beta1 in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor beta (RARbeta), hepatocyte growth factor (HGF), cellular retinoic acid-binding protein II (CRABPII), vimentin, E-cadherin, Snail, and beta-catenin was assessed by immunoblotting. The cellular localization of vimentin and beta-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1458 genes after C-peptide exposure for 18 h or 48 h, respectively. From these, members of the antifibrotic retinoic acid (RA)- and HGF-signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARbeta, CRABPII, and HGF. We confirmed a role for RA in reversal of TGF-beta1-induced changes associated with epithelial-mesenchymal transition, including expression changes in Snail, E-cadherin, vimetin, and redistribution of beta-catenin. Importantly, these TGF-beta1-induced changes were inhibited by C-peptide. Further, effects of TGF-beta1 on Snail and E-cadherin expression were blocked by HGF, and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA-signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy.
Journal of the American Society of Nephrology, 2006
Cell loss by apoptosis occurs in renal injury such as diabetic nephropathy. TNF-␣ is a cytokine t... more Cell loss by apoptosis occurs in renal injury such as diabetic nephropathy. TNF-␣ is a cytokine that induces apoptosis and has been implicated in the pathogenesis of diabetic nephropathy. The aim was to investigate whether C-peptide or insulin could modulate TNF-␣-mediated cell death in opossum kidney proximal tubular cells and to examine the mechanism(s) of any effects observed. C-peptide and insulin protect against TNF-␣-induced proximal tubular cell toxicity and apoptosis. Cell viability was analyzed by methylthiazoletetrazolium assay; cell viability was reduced to 60.8 ؎ 2.7% of control after stimulation with 300 ng/ml TNF-␣. Compromised cell viability was reversed by pretreatment with 5 nM C-peptide or 100 nM insulin. TNF-␣-induced apoptosis was detected by DNA nick-end labeling and by measuring histone associated DNA fragments using ELISA. By ELISA assay, 300 ng/ml TNF-␣ increased apoptosis by 145.8 ؎ 4.9% compared with controls, whereas 5 nM C-peptide and 100 nM insulin reduced apoptosis to 81.6 ؎ 4.8 and 77.4 ؎ 3.1% of control, respectively. The protective effects of C-peptide and insulin were associated with activation of NF-B. Activation of NF-B by C-peptide was pertussis toxin sensitive and dependent on activation of G␣ i . Phosphatidylinositol 3-kinase but not extracellular signal regulated mitogen-activated protein kinase mediated C-peptide and insulin activation of NF-B. The cytoprotective effects of both C-peptide and insulin were related to increased expression of TNF receptor-associated factor 2, the product of an NF-Bdependent survival gene. These data suggest that C-peptide and/or insulin activation of NF-B-regulated survival genes protects against TNF-␣-induced renal tubular injury in diabetes. The data further support the concept of C-peptide as a peptide hormone in its own right and suggest a potential therapeutic role for C-peptide.
Stimulation of gonadotropin-releasing hormone (GnRH) receptors with the GnRH analogue buserelin e... more Stimulation of gonadotropin-releasing hormone (GnRH) receptors with the GnRH analogue buserelin enhances expression of the zinc finger transcription factor Egr-1 in a pituitary gonadotroph cell line. The signaling cascade is blocked by overexpression of MAP kinase phosphatase-1 that dephosphorylates extracellular signal-regulated protein kinase in the nucleus. Chromatin immunoprecipitation experiments revealed that the phosphorylated form of Elk-1, a key regulator of gene transcription driven by serum response element (SRE), binds to the 5'-upstream region of the Egr-1 gene in buserelin-stimulated gonadotrophs. Expression of a dominant-negative mutant of Elk-1 completely blocked Egr-1 expression, indicating that Elk-1 connects the intracellular signaling cascade elicited by activation of GnRH receptors with transcription of the Egr-1 gene. GnRH receptor activation additionally induced the phosphorylation of CREB, which in its phosphorylated form bound to the Egr-1 gene. Expression of a dominant-negative mutant of CREB reduced GnRH receptor-induced upregulation of Egr-1 expression, indicating that CREB plays a role in the signaling pathway that regulates Egr-1 expression in gonadotrophs. We further identified the genes encoding basic fibroblast growth factor, tumor necrosis factor alpha, and transforming growth factor beta as bona fide target genes of Egr-1 in gonadotrophs. The analysis of gonadotroph cells that express--in addition to GnRH receptors--muscarinic M(3) acetylcholine receptors revealed that the nuclear events connecting GnRH receptors and muscarinic M(3) acetylcholine receptors with the Egr-1 gene are indistinguishable.
Peroxisome proliferator-activated receptor ␥ (PPAR␥) has key roles in the regulation of adipogene... more Peroxisome proliferator-activated receptor ␥ (PPAR␥) has key roles in the regulation of adipogenesis, inflammation, and lipid and glucose metabolism. C-peptide is believed to be inert and without appreciable biological functions. Recent studies suggest that C-peptide possesses multiple functions. The present study investigated the effects of insulin and C-peptide on PPAR␥ transcriptional activity in opossum kidney proximal tubular cells. Both insulin and C-peptide induced a concentration-dependent stimulation of PPAR␥ transcriptional activity. Both agents substantially augmented thiazolidinedione-stimulated PPAR␥ transcriptional activity. Neither insulin nor C-peptide had any effect on the expression levels of PPAR␥. GW9662, a PPAR␥ antagonist, blocked PPAR␥ activation by thiazolidinediones but had no effect on either insulin-or C-peptide-stimulated PPAR␥ transcriptional activity. Co-transfection of opossum kidney cells with dominant negative mitogen-activated protein kinase kinase significantly depressed basal PPAR␥ transcriptional activity but had no effect on that induced by either insulin or C-peptide. Both insulin-and C-peptide-stimulated PPAR␥ transcriptional activity were attenuated by wortmannin and by expression of a dominant negative phosphatidylinositol (PI) 3-kinase p85 regulatory subunit. In addition PI 3-kinase-dependent phosphorylation of PPAR␥ was observed after stimulation by C-peptide or insulin. C-peptide effects but not insulin on PPAR␥ transcriptional activity were abolished by pertussis toxin pretreatment. Finally both C-peptide and insulin positively control the expression of the PPAR␥-regulated CD36 scavenger receptor in human THP-1 monocytes. We concluded that insulin and C-peptide can stimulate PPAR␥ activity in a ligand-independent fashion and that this effect is mediated by PI 3-kinase. These results support a new and potentially important physiological role for C-peptide in regulation of PPAR␥-related cell functions.
We investigated the role played by agonist-mediated phosphorylation of the G(q/11)-coupled M(3)-m... more We investigated the role played by agonist-mediated phosphorylation of the G(q/11)-coupled M(3)-muscarinic receptor in the mechanism of activation of the mitogen-activated protein kinase pathway, ERK-1/2, in transfected Chinese hamster ovary cells. A mutant of the M(3)-muscarinic receptor, where residues Lys(370)-Ser(425) of the third intracellular loop had been deleted, showed a reduced ability to activate the ERK-1/2 pathway. This reduction was evident despite the fact that the receptor was able to couple efficiently to the phospholipase C second messenger pathway. Importantly, the ERK-1/2 responses to both the wild-type M(3)-muscarinic receptor and DeltaLys(370)-Ser(425) receptor mutant were dependent on the activity of protein kinase C. Our results, therefore, indicate the existence of two mechanistic components to the ERK-1/2 response, which appear to act in concert. First, the activation of protein kinase C through the diacylglycerol arm of the phospholipase C signaling pathway and a second component, absent in the DeltaLys(370)-Ser(425) receptor mutant, that is independent of the phospholipase C signaling pathway. The reduced ability of the DeltaLys(370)-Ser(425) receptor mutant to activate the ERK-1/2 pathway correlated with an approximately 80% decrease in the ability of the receptor to undergo agonist-mediated phosphorylation. Furthermore, we have previously shown that M(3)-muscarinic receptor phosphorylation can be inhibited by a dominant negative mutant of casein kinase 1alpha and by expression of a peptide corresponding to the third intracellular loop of the M(3)-muscarinic receptor. Expression of these inhibitors of receptor phosphorylation reduced the wild-type M(3)-muscarinic receptor ERK-1/2 response. We conclude that phosphorylation of the M(3)-muscarinic receptor on sites in the third intracellular loop by casein kinase 1alpha contributes to the mechanism of receptor activation of ERK-1/2 by working in concert with the diacylglycerol/PKC arm of the phospholipase C signaling pathway.
In this study we have quantitatively assessed the basal turnover of phosphatidylinositol 4,5-bisp... more In this study we have quantitatively assessed the basal turnover of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and M3-muscarinic receptor-mediated changes in phosphoinositides in the human neuroblastoma cell line, SH-SY5Y. We demonstrate that the polyphosphoinositides represent a minor fraction of the total cellular phosphoinositide pool and that in addition to rapid, sustained increases in [3H]inositol phosphates dependent upon the extent of receptor activation by carbachol, there are equally rapid and sustained reductions in the levels of polyphosphoinositides. Compared with phosphatidylinositol 4-phosphate (PtdIns(4)P), PtdIns(4,5)P2 was reduced with less potency by carbachol and recovered faster following agonist removal suggesting protection of PtdIns(4,5)P2 at the expense of PtdIns(4)P and indicating specific regulatory mechanism(s). This does not involve a pertussis toxin-sensitive G-protein regulation of PtdIns(4)P 5-kinase. Using wortmannin to inhibit PtdIns 4-kinase activity, we demonstrate that the immediate consequence of blocking the supply of PtdIns(4)P (and therefore PtdIns(4,5)P2) is a failure of agonist-mediated phosphoinositide and Ca2+ signaling. The use of wortmannin also indicated that PtdIns is not a substrate for receptor-activated phospholipase C and that 15% of the basal level of PtdIns(4,5)P2 is in an agonist-insensitive pool. We estimate that the agonist-sensitive pool of PtdIns(4,5)P2 turns over every 5 s (0.23 fmol/cell/min) during sustained receptor activation by a maximally effective concentration of carbachol. Immediately following agonist addition, PtdIns(4,5)P2 is consumed >3 times faster (0.76 fmol/cell/min) than during sustained receptor activation which represents, therefore, utilization by a partially desensitized receptor. These data indicate that resynthesis of PtdIns(4,5)P2 is required to allow full early and sustained phases of receptor signaling. Despite the critical dependence of phosphoinositide and Ca2+ signaling on PtdIns(4,5)P2 resynthesis, we find no evidence that this rate resynthesis is limiting for agonist-mediated responses.
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Papers by Gary Willars