MCP-1 Upregulates Amylin Expression in Murine Pancreatic β Cells through ERK/JNK-AP1 and NF-κB Related Signaling Pathways Independent of CCR2

BACKGROUND: Amylin is the most abundant component of islet amyloid implicated in the development of type 2 diabetes. Plasma amylin levels are elevated in individuals with obesity and insulin resistance. Monocyte chemoattractant protein-1 (MCP-1, CCL2) is involved in insulin resistance of obesity and type 2 diabetes. We investigated the effect of MCP-1 on amylin expression and the underlying mechanisms with murine pancreatic β-cell line MIN6 and pancreatic islets. METHODOLOGY/PRINCIPAL FINDINGS: We found that MCP-1 induced amylin expression at transcriptional level and increased proamylin and intermediate forms of amylin at protein level in MIN6 cells and islets. However, MCP-1 had no effect on the expressions of proinsulin 1 and 2, as well as prohormone convertase (PC) 1/3 and PC2, suggesting that MCP-1 specifically induces amylin expression in β-cells. Mechanistic studies showed that although there is no detectable CCR2 mRNA in MIN6 cells and islets, pretreatment of MIN6 cells with pertussis toxin inhibited MCP-1 induced amylin expression, suggesting that alternative Gi-coupled receptor(s) mediates the inductive effect of MCP-1. MCP-1 rapidly induced ERK1/2 and JNK phosphorylation. Inhibitors for MEK1/2 (PD98059), JNK (SP600125) or AP1 (curcumin) significantly inhibited MCP-1-induced amylin mRNA expression. MCP-1 failed to induce amylin expression in pancreatic islets isolated from Fos knockout mice. EMSA showed that JNK and ERK1/2 were involved in MCP-1-induced AP1 activation. These results suggest that MCP-1 induces murine amylin expression through AP1 activation mediated by ERK1/2 or JNK. Further studies showed that treatment of MIN6 cells with NF-κB inhibitor or overexpression of IκBα dominant-negative construct in MIN6 cells significantly inhibited MCP-1-induced amylin expression, suggesting that NF-κB related signaling also participates in MCP-1-induced murine amylin expression. CONCLUSIONS/SIGNIFICANCE: MCP-1 induces amylin expression through ERK1/2/JNK-AP1 and NF-κB related signaling pathways independent of CCR2. Amylin upregulation by MCP-1 may contribute to elevation of plasma amylin in obesity and insulin resistance

Renal HIV Expression Is Unaffected by Serum LPS Levels in an HIV Transgenic Mouse Model of LPS Induced Kidney Injury

Acute kidney injury (AKI) is associated with increased rates of mortality. For unknown reasons, HIV infected individuals have a higher risk of AKI than uninfected persons. We tested our hypothesis that increased circulating LPS increases renal expression of HIV and that HIV transgenic (Tg26) mice have increased susceptibility to AKI. Tg26 mice harbor an HIV transgene encoding all HIV genes except gag and pol, and develop a phenotype analogous to HIVAN. Mice were used at 4–6 weeks of age before the onset of gross renal disease. Mice were injected i.p. with LPS or sterile saline. Renal function, tubular injury, cytokine expression, and HIV transcription were evaluated in Tg26 and wild type (WT) mice. LPS injection induced a median 60.1-fold increase in HIV expression in spleen but no change in kidney. There was no significant difference in renal function, cytokine expression, or tubular injury scores at baseline or 24 hours after LPS injection. HIV transcription was also analyzed in vitro using a human renal tubular epithelial cell (RTEC) line. HIV transcription increased minimally in human RTEC, by 1.47 fold, 48 hours after LPS exposure. We conclude that Tg26 mice do not increase HIV expression or have increased susceptibility to LPS induced AKI. The increased risk of AKI in HIV infected patients is not mediated via increased renal expression of HIV in the setting of sepsis. Moreover, renal regulation of HIV transcription is different to that in the spleen

Aldosterone potentiates angiotensin II-induced signaling in vascular smooth muscle cells

Background - In a double-transgenic human renin and human angiotensinogen rat model, we found that mineralocorticoid receptor (MR) blockade ameliorated angiotensin II (Ang II)-induced renal and cardiac damage. How Ang II and aldosterone (Ald) might interact is ill defined. Methods and Results - We investigated the effects of Ang II (10-7 mol/L) and Ald (10 -7 mol/L) on extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling in vascular smooth muscle cells (VSMCs) with Western blotting and confocal microscopy. Ang II induced ERK 1/2 and JNK phosphorylation by 2 minutes. Ald achieved the same at 10 minutes. Ang II + Ald had a potentiating effect by 2 minutes. Two oxygen radical scavengers and the epidermal growth factor receptor (EGFR) antagonist AG1478 reduced Ang II-, Ald-, and combination-induced ERK1/2 phosphorylation. Preincubating the cells with the MR blocker spironolactone (10-6 mol/L) abolished Ang II-induced ROS generation, EGFR transactivation, and ERK1/2 phosphorylation. Conclusions - Ald potentiates Ang II-induced ERK-1/2 and JNK phosphorylation. Oxygen radicals, the MR, and the EGFR play a role in early signaling induced by Ang II and Ald in VSMCs. These in vitro data may help explain the effects of MR blockade on Ang II-induced end-organ damage in vivo

AT1 receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase

BACKGROUND: We recently identified agonistic autoantibodies directed against the angiotensin AT1 receptor (AT1-AA) in the plasma of preeclamptic women. To elucidate their role further, we studied the effects of AT1-AA on reactive oxygen species (ROS), NADPH oxidase expression, and nuclear factor-kappaB (NF-kappaB) activation. METHODS AND RESULTS: We investigated human vascular smooth muscle cells (VSMC) and trophoblasts, as well as placentas. AT1-AA were isolated from sera of preeclamptic women. Angiotensin II (Ang II) and AT1-AA increased ROS production and the NADPH oxidase components, p22, p47, and p67 phox in Western blotting. We next tested if AT1-AA lead to NF-kappaB activation in VSMC and trophoblasts. AT1-AA activated NF-kappaB. Inhibitor-kappaBalpha (I-kappaBalpha) expression was reduced in response to AT1-AA. AT1 receptor blockade with losartan, diphenylene iodonium, tiron, and antisense against p22 phox all reduced ROS production and NF-kappaB activation. VSMC from p47phox-/- mice showed markedly reduced ROS generation and NF-kappaB activation in response to Ang II and AT1-AA. The p22, p47, and p67 phox expression in placentas from preeclamptic patients was increased, compared with normal placentas. Furthermore, NF-kappaB was activated and I-kappaBalpha reduced in placentas from preeclamptic women. CONCLUSIONS: NADPH oxidase is potentially an important source of ROS that may upregulate NF-kappaB in preeclampsia. We suggest that AT1-AA through activation of NADPH oxidase could contribute to ROS production and inflammatory responses in preeclampsia