Michael C. Lawrence

The roles of MAPKs in disease

MAP kinases transduce signals that are involved in a multitude of cellular pathways and functions in response to a variety of ligands and cell stimuli. Aberrant or inappropriate functions of MAPKs have now been identified in diseases ranging from cancer to inflammatory disease to obesity and diabetes. In many cell types, the MAPKs ERK1/2 are linked to cell proliferation. ERK1/2 are thought to play a role in some cancers, because mutations in Ras and B-Raf, which can activate the ERK1/2 cascade, are found in many human tumors. Abnormal ERK1/2 signaling has also been found in polycystic kidney disease, and serious developmental disorders such as cardio-facio-cutaneous syndrome arise from mutations in components of the ERK1/2 cascade. ERK1/2 are essential in well-differentiated cells and have been linked to long-term potentiation in neurons and in maintenance of epithelial polarity. Additionally, ERK1/2 are important for insulin gene transcription in pancreatic beta cells, which produce insulin in response to increases in circulating glucose to permit efficient glucose utilization and storage in the organism. Nutrients and hormones that induce or repress insulin secretion activate and/or inhibit ERK1/2 in a manner that reflects the secretory demand on beta cells. Disturbances in this and other regulatory pathways may result in the contribution of ERK1/2 to the etiology of certain human disorders.

Regulation of ERK1 and ERK2 by Glucose and Peptide Hormones in Pancreatic β Cells

We showed previously that ERK1/2 were activated by glucose and amino acids in pancreatic β cells. Here we examine and compare signaling events that are necessary for ERK1/2 activation by glucose and other stimuli in β cells. We find that agents that interrupt Ca2+ signaling by a variety of mechanisms interfere with glucose- and glucagon-like peptide (GLP-1)-stimulated ERK1/2 activity. In particular, calmodulin antagonists, FK506, and cyclosporin, immunosuppressants that inhibit the calcium-dependent phosphatase calcineurin, suppress ERK1/2 activation by both glucose and GLP-1. Ca2+ signaling from intracellular stores is also essential for ERK1/2 activation, because thapsigargin blocks ERK1/2 activation by glucose or GLP-1. The glucose-sensitive mechanism is distinct from that used by phorbol ester or insulin to stimulate ERK1/2 but shares common features with that used by GLP-1.

The protein kinases ERK1/2 and their roles in pancreatic beta cells.

Extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2) activities are modulated in a manner that reflects the secretory demand on β cells to integrate long‐ and short‐term nutrient sensing information. Our studies have focused on the mechanisms of ERK1/2 activation in β cells and on the actions of ERK1/2 that regulate β cell function. Insulin and growth factors regulate ERK1/2 in β cells in a largely calcium‐independent manner. Nutrients and anticipatory hormones, in contrast, activate ERK1/2 in a calcium‐dependent manner in these cells. We are exploring the key intermediates in these distinct activation pathways and find that calcineurin is essential for the nutrient pathway but is not essential for the growth factor pathway. Using reporter assays, heterologous reconstitution, electrophoretic mobility shift assays, Northern analysis, Q‐PCR and chromatin immunoprecipitation, we have examined several genes that are regulated by ERK1/2, primarily the insulin gene and the apoptotic factor C/EBP‐homologous protein (CHOP)‐10 (GADD153/DDIT‐3), a bZIP protein. ERK1/2‐sensitive transcriptional regulators common to these two genes are C/EBP‐β and MafA. The insulin promoter is both positively and negatively regulated by glucose and other nutrients. Exposure to glucose for minutes to hours causes an increase in the rate of insulin gene transcription. In contrast, exposure to elevated glucose for 48 h or more results in inhibition of the insulin gene promoter. Both of these processes depend on ERK1/2 activity. Expression of CHOP is induced by stresses including nutrient deprivation and endoplasmic reticulum stress. CHOP gene expression, especially that regulated by nutrients, is also ERK1/2‐dependent in β cells, These studies support the hypothesis that the genes regulated by ERK1/2 and the mechanisms employed are key to maintaining normal β cell function.

Chromatin-bound mitogen-activated protein kinases transmit dynamic signals in transcription complexes in β-cells

MAPK pathways regulate transcription through phosphorylation of transcription factors and other DNA-binding proteins. In pancreatic β-cells, ERK1/2 are required for transcription of the insulin gene and several other genes in response to glucose. We show that binding of glucose-sensitive transcription activators and repressors to the insulin gene promoter depends on ERK1/2 activity. We also find that glucose and NGF stimulate the binding of ERK1/2 to the insulin gene and other promoters. An ERK1/2 cascade module, including MEK1/2 and Rsk, are found in complexes bound to these promoters. These findings imply that MAPK-containing signaling complexes are positioned on sensitive promoters with their protein substrates to modulate transcription in situ in response to incoming signals.

Withaferin A inhibits pro-inflammatory cytokine-induced damage to islets in culture and following transplantation

Aims/hypothesisBeta cell death triggered by pro-inflammatory cytokines plays a central role in the pathogenesis of type 1 diabetes and loss of transplanted islets. The nuclear factor κB (NF-κB) signalling pathway is a key regulator of beta cell stress response, survival and apoptosis. Withaferin A (WA), a steroidal lactone derived from Withania somnifera, has been demonstrated to be a potent, safe, anti-inflammatory molecule that can inhibit NF-κB signalling. Therefore, we evaluated the ability of WA to protect mouse and human islets from the damaging effects of pro-inflammatory cytokines in vitro and following intraportal transplantation.MethodsMouse and human islets were treated with a cytokine cocktail, and NF-κB activation was measured by immunoblots, p65 nuclear translocation and chromatin immunoprecipitation of p65-bound DNA. Intraportal transplantation of a marginal mass of syngeneic mouse islets was performed to evaluate the in vivo protective effect of WA.ResultsTreatment with WA substantially improved islet engraftment of syngeneic islets (83% for infusion with 200 islets + WA; 0% for 200 islets + vehicle) in a mouse model of diabetes, compared with marginal graft controls with superior islet function in WA-treated mice confirmed by glucose tolerance test. Treatment of human and mouse islets with WA prevented cytokine-induced cell death, inhibited inflammatory cytokine secretion and protected islet potency.ConclusionsWA was shown to be a strong inhibitor of the inflammatory response in islets, protecting against cytokine-induced cell damage while improving survival of transplanted islets. These results suggest that WA could be incorporated as an adjunctive treatment to improve islet transplant outcome.

Inflammatory Response in Islet Transplantation

Islet cell transplantation is a promising beta cell replacement therapy for patients with brittle type 1 diabetes as well as refractory chronic pancreatitis. Despite the vast advancements made in this field, challenges still remain in achieving high frequency and long-term successful transplant outcomes. Here we review recent advances in understanding the role of inflammation in islet transplantation and development of strategies to prevent damage to islets from inflammation. The inflammatory response associated with islets has been recognized as the primary cause of early damage to islets and graft loss after transplantation. Details on cell signaling pathways in islets triggered by cytokines and harmful inflammatory events during pancreas procurement, pancreas preservation, islet isolation, and islet infusion are presented. Robust control of pre- and peritransplant islet inflammation could improve posttransplant islet survival and in turn enhance the benefits of islet cell transplantation for patients who are insulin dependent. We discuss several potent anti-inflammatory strategies that show promise for improving islet engraftment. Further understanding of molecular mechanisms involved in the inflammatory response will provide the basis for developing potent therapeutic strategies for enhancing the quality and success of islet transplantation.

Differential regulation of CHOP-10/GADD153 gene expression by MAPK signaling in pancreatic β-cells

CHOP-10 (GADD153/DDIT-3) is a bZIP protein involved in differentiation and apoptosis. Its expression is induced in response to stresses such as nutrient deprivation, perturbation of the endoplasmic reticulum, redox imbalance, and UV exposure. Here we show that CHOP expression is induced in cultured pancreatic β-cells maintained in a basal glucose concentration of 5.5 mM and repressed by stimulatory glucose (≥11 mM). Both induction and repression of CHOP are dependent on the MAPKs ERK1 and ERK2. Two regulatory composite sites containing overlapping MafA response elements (MARE) and CAAT enhancer binding (CEB) elements regulate transcription in an ERK1/2-dependent manner. One site (MARE-CEB), from −320 to −300 bp in the promoter, represses transcription. The other site (CEB-MARE), from +2,628 to +2,641 bp in the first intron of the CHOP gene, activates it. MafA can influence transcription of both sites. The MARE-CEB is repressed by MafA, whereas the CEB-MARE site, which is homologous to the A2C1 component of the glucose-sensitive RIPE3b region of the insulin gene promoter, is activated by MafA. These results indicate that ERK1/2 have dual roles in regulating CHOP gene expression via both promoter and intronic regions, depending on environmental and metabolic stresses imposed on pancreatic β-cells.

Evaluation of Microrna375 as a Novel Biomarker for Graft Damage in Clinical Islet Transplantation

Background Early and sensitive detection of islet graft damage is essential for improving posttransplant outcomes. MicroRNA 375 (miR375) has been reported as a biomarker of pancreatic &bgr;-cell death in small animal models. Methods The miR375 levels were measured in purified human islets, sera from patients with autologous and allogeneic islet transplantation as well as total pancreatectomy alone (nontransplanted group). The miR375 levels were also determined in a miniaturized in vitro tube model comprising human islets and autologous blood. Results The miR375 expression level in islets was dose-dependent (P < 0.001) and significantly elevated after islet damage in plasma in the in vitro model (P = 0.003). Clinical analysis revealed that circulating miR375 levels in both autologous and allogeneic islet recipients were significantly elevated for 7 days after islet infusion, compared with the nontransplanted group (P = 0.005 and <0.001, respectively). Furthermore, miR375 detected the difference in islet graft damage among 3 different anti-inflammatory protocols for clinical autologous transplantation (P < 0.01). Conclusions Circulating miR375 can be a reliable biomarker to detect graft damage in clinical islet transplantation because serum C-peptide and proinsulin levels are difficult to interpret due to the influence of multiple factors, such as &bgr;-cell stress and physiological response.

Multiple chromatin-bound protein kinases assemble factors that regulate insulin gene transcription

During the onset of diabetes, pancreatic β cells become unable to produce sufficient insulin to maintain blood glucose within the normal range. Proinflammatory cytokines have been implicated in impaired β cell function. To understand more about the molecular events that reduce insulin gene transcription, we examined the effects of hyperglycemia alone and together with the proinflammatory cytokine interleukin-1β (IL-1β) on signal transduction pathways that regulate insulin gene transcription. Exposure to IL-1β in fasting glucose activated multiple protein kinases that associate with the insulin gene promoter and transiently increased insulin gene transcription in β cells. In contrast, cells exposed to hyperglycemic conditions were sensitized to the inhibitory actions of IL-1β. Under these conditions, IL-1β caused the association of the same protein kinases, but a different combination of transcription factors with the insulin gene promoter and began to reduce transcription within 2 h; stimulatory factors were lost, RNA polymerase II was lost, and inhibitory factors were bound to the promoter in a kinase-dependent manner.

MAP kinases and their roles in pancreatic β-cells

We discuss our work examining regulation and functions of mitogen-activated protein kinases, particularly ERK1 and ERK2, in pancreatic β-cells. These enzymes are activated by glucose, other nutrients, and insulinogenic hormones. Their activation by these agents is calcium-dependent. A number of other stimuli also activate ERK1/2, but by mechanisms distinct from those involved in nutrient sensing. Inhibition of ERK1/2 has no apparent effect on insulin secretion measured after 2 h. On the other hand, ERK1/2 activity is required for maximal glucose-dependent activation of the insulin gene promoter. The primary effort has focused on INS-1 cell lines, with supporting and confirmatory studies in intact islets and other β-cell lines, indicating the generality of our findings in β-cell function. Thus ERK1/2 participate in transmitting glucose-sensing information to β-cell functions. These kinases most likely act directly and indirectly on multiple pathways that regulate β-cell function and, in particular, to transduce an elevated glucose signal into insulin gene transcription.