Pioglitazone inhibits advanced glycation end product-induced matrix metalloproteinases and apoptosis by suppressing the activation of MAPK and NF-κB

Apoptosis and degeneration coming mainly from chondrocytes are important mechanisms in the onset and progression of osteoarthritis. Specifically, advanced glycation end products (AGEs) play an important role in the pathogenesis of osteoarthritis. Pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist has a protective effect on cartilage. This study aims to evaluate the effect of pioglitazone on AGEs-induced chondrocyte apoptosis and degeneration and their underlying mechanism. The in vitro study shows that AGEs induce cleavage of caspase-3 and PARP, up-regulate MMP-13 expression, enhance chondrocyte apoptosis and down-regulate PPARγ expression in human primary chondrocytes, which is reversed by pioglitazone. Furthermore, AGEs activate phosphorylation of Erk, JNK, and p38, and pioglitazone reverses AGEs-induced phosphorylation of Erk and p38. AGEs-induced degradation of IκBα and translocation of nuclear NF-κB p65 is reversed by pioglitazone. Pretreatment of chondrocytes with SB202190 (p38 inhibitor), SP600125 (JNK inhibitor) and BAY-11-7082 (NF-κB inhibitor) inhibit AGEs-induced apoptosis and degeneration. In vivo experiments suggest that pioglitazone reverses AGEs-induced cartilage degeneration and apoptosis in a mouse model, as demonstrated by HE and Safranin O staining, immunohistochemical analyses of Type II collagen (Col II), metalloproteinases (MMPs) and caspase-3. These findings suggest that pioglitazone, a PPARγ agonist, inhibits AGEs-induced chondrocytes apoptosis and degeneration via suppressing the activation of MAPK and NF-κB.     

The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1α expression

Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 alpha (PGC-1α) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1α in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1α expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1α mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1α expression while stimulating VSMC migration. Mechanistic study showed that suppression of PGC-1α by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1α had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1α decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPARγ activation by a PPARγ antagonist GW9662 abolished the suppressive effects of PGC-1α on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1α were enhanced by a PPARγ agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1α expression. PGC-1α suppresses PDGF-induced VSMC migration through PPARγ coactivation and, consequently, p38 MAPK inhibition.     

Pioglitazone upregulates adiponectin receptor 2 in 3T3-L1 adipocytes

AimsPioglitazone, a full peroxisome proliferator-activated receptor (PPAR)-γ agonist, improves insulin sensitivity by increasing circulating adiponectin levels. However, the molecular mechanisms by which pioglitazone induces insulin sensitization are not fully understood. In this study, we investigated whether pioglitazone improves insulin resistance via upregulation of either 2 distinct receptors for adiponectin (AdipoR1 or AdipoR2) expression in 3T3-L1 adipocytes.Main methodsGlucose uptake was evaluated by 2-[³H] deoxy-glucose uptake assay in 3T3-L1 adipocytes with pioglitazone treatment. AdipoR1 and AdipoR2 mRNA expressions were analyzed by qRT–PCR.Key findingsWe first confirmed that pioglitazone significantly increased insulin-induced 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes. Next, we investigated the mRNA expression and regulation of AdipoR1 and AdipoR2 after treatment with pioglitazone. Interestingly, pioglitazone significantly induced AdipoR2 expression but it did not affect AdipoR1 expression. In addition, adenovirus-mediated PPARγ expression significantly enhanced the effects of pioglitazone on insulin-stimulated 2-DOG uptake and AdipoR2 expression in 3T3-L1 adipocytes. These data suggest that pioglitazone enhances adiponectin's autocrine and paracrine actions in 3T3-L1 adipocytes via upregulation of PPARγ-mediated AdipoR2 expression. Furthermore, we found that pioglitazone significantly increased AMP-activated protein kinase (AMPK) phosphorylation in insulin-stimulated 3T3-L1 adipocytes, but it did not lead to the phosphorylation of IRS-1, Akt, or protein kinase Cλ/ζ.SignificanceOur results suggest that pioglitazone increases insulin sensitivity, at least partly, by PPARγ-AdipoR2-mediated AMPK phosphorylation in 3T3-L1 adipocytes. In conclusion, the upregulation of AdipoR2 expression may be one of the mechanisms by which pioglitazone improves insulin resistance in 3T3-L1 adipocytes.     

Peroxisome Proliferator-activated receptor γ activation by ligands and dephosphorylation induces proprotein convertase subtilisin kexin type 9 and low density lipoprotein receptor expression

Proprotein convertase subtilisin kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis by enhancing the degradation of LDL receptor (LDLR) protein. Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to be atheroprotective. PPARγ can be activated by ligands and/or dephosphorylation with ERK1/2 inhibitors. The effect of PPARγ on PCSK9 and LDLR expression remains unknown. In this study, we investigated the effects of PPARγ on PCSK9 and LDLR expression. At the cellular levels, PPARγ ligands induced PCSK9 mRNA and protein expression in HepG2 cells. PCSK9 expression was induced by inhibition of ERK1/2 activity but inhibited by ERK1/2 activation. The mutagenic study and promoter activity assay suggested that the induction of PCSK9 expression by ERK1/2 inhibitors was tightly linked to PPARγ dephosphorylation. However, PPARγ activation by ligands or ERK1/2 inhibitors induced hepatic LDLR expression. The promoter assay indicated that the induction of LDLR expression by PPARγ was sterol regulatory element-dependent because PPARγ enhanced sterol regulatory element-binding protein 2 (SREBP2) processing. In vivo, administration of pioglitazone or U0126 alone increased PCSK9 expression in mouse liver but had little effect on PCSK9 secretion. However, the co-treatment of pioglitazone and U0126 enhanced both PCSK9 expression and secretion. Similar to in vitro, the increased PCSK9 expression by pioglitazone and/or U0126 did not result in decreased LDLR expression and function. In contrast, pioglitazone and/or U0126 increased LDLR protein expression and membrane translocation, SREBP2 processing, and CYP7A1 expression in the liver, which led to decreased total and LDL cholesterol levels in serum. Our results indicate that although PPARγ activation increased PCSK9 expression, PPARγ activation induced LDLR and CYP7A1 expression that enhanced LDL cholesterol metabolism.     

RAGE mediates accelerated diabetic vein graft atherosclerosis induced by combined mechanical stress and AGEs via synergistic ERK activation

Aims/Hypothesis

Diabetes with hypertension rapidly accelerates vascular disease, but the underlying mechanism remains unclear. We evaluated the hypothesis that the receptor of advanced glycation end products (RAGE) might mediate combined signals initiated by diabetes-related AGEs and hypertension-induced mechanical stress as a common molecular sensor.

Methods

In vivo surgical vein grafts created by grafting vena cava segments from C57BL/6J mice into the common carotid arteries of streptozotocin (STZ)-treated and untreated isogenic mice for 4 and 8 weeks were analyzed using morphometric and immunohistochemical techniques. In vitro quiescent mouse vascular smooth muscle cells (VSMCs) with either knockdown or overexpression of RAGE were subjected to cyclic stretching with or without AGEs. Extracellular signal-regulated kinase (ERK) phosphorylation and Ki-67 expression were investigated.

Results

Significant increases in neointimal formation, AGE deposition, Ki-67 expression, and RAGE were observed in the vein grafts of STZ-induced diabetic mice. The highest levels of ERK phosphorylation and Ki-67 expression in VSMCs were induced by simultaneous stretch stress and AGE exposure. The synergistic activation of ERKs and Ki-67 in VSMCs was significantly inhibited by siRNA-RAGE treatment and enhanced by over-expression of RAGE.

Conclusion

RAGE may mediate synergistically increased ERK activation and VSMC proliferation induced by mechanical stretching with and without AGEs. It may serve as a common molecular bridge between the two, accelerating vascular remodeling. This study provides potential drug targets and novel therapeutic strategies for the treatment of vascular diseases resulting from diabetes with hypertension.     

PDGF-induced vascular smooth muscle cell proliferation is associated with dysregulation of insulin receptor substrates

In vascular smooth muscle cells (VSMCs), platelet-derived growth factor (PDGF) plays a major role in inducing phenotypic switching from contractile to proliferative state. Importantly, VSMC phenotypic switching is also determined by the phosphorylation state/expression levels of insulin receptor substrate (IRS), an intermediary signaling component that is shared by insulin and IGF-I. To date, the roles of PDGF-induced key proliferative signaling components including Akt, p70S6kinase, and ERK1/2 on the serine phosphorylation/expression of IRS-1 and IRS-2 isoforms remain unclear in VSMCs. We hypothesize that PDGF-induced VSMC proliferation is associated with dysregulation of insulin receptor substrates. Using human aortic VSMCs, we demonstrate that prolonged PDGF treatment led to sustained increases in the phosphorylation of protein kinases such as Akt, p70S6kinase, and ERK1/2, which mediate VSMC proliferation. In addition, PDGF enhanced IRS-1/IRS-2 serine phosphorylation and downregulated IRS-2 expression in a time- and concentration-dependent manner. Notably, phosphoinositide 3-kinase (PI 3-kinase) inhibitor (PI-103) and mammalian target of rapamycin inhibitor (rapamycin), which abolished PDGF-induced Akt and p70S6kinase phosphorylation, respectively, blocked PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. In contrast, MEK1/ERK inhibitor (U0126) failed to block PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. PDGF-induced IRS-2 downregulation was prevented by lactacystin, an inhibitor of proteasomal degradation. Functionally, PDGF-mediated IRS-1/IRS-2 dysregulation resulted in the attenuation of insulin-induced IRS-1/IRS-2-associated PI 3-kinase activity. Pharmacological inhibition of PDGF receptor tyrosine kinase with imatinib prevented IRS-1/IRS-2 dysregulation and restored insulin receptor signaling. In conclusion, strategies to inhibit PDGF receptors would not only inhibit neointimal growth but may provide new therapeutic options to prevent dysregulated insulin receptor signaling in VSMCs in nondiabetic and diabetic states.     

PPARγ Agonists Attenuate Palmitate-Induced ER Stress through Up-Regulation of SCD-1 in Macrophages

Background

Clinical trials have shown that treatment of patients with type 2 diabetes with pioglitazone, a peroxisome proliferator-activated receptor (PPAR)γ agonist, reduces cardiovascular events. However, the effect of PPARγ agonists on endoplasmic reticulum (ER) stress that plays an important role in the progression of atherosclerosis has not been determined. We sought to determine the effect of PPARγ agonists on ER stress induced by palmitate, the most abundant saturated fatty acid in the serum.

Methods and Results

Protein expression of ER stress marker was evaluated by Western blot analysis and stearoyl-CoA desaturase1 (SCD-1) mRNA expression was evaluated by qRT-PCR. Macrophage apoptosis was detected by flowcytometry. Pioglitazone and rosiglitazone reduced palmitate-induced phosphorylation of PERK, a marker of ER stress, in RAW264.7, a murine macrophage cell line. Pioglitazone also suppressed palmitate-induced apoptosis in association with inhibition of CHOP expression, JNK phosphorylation and cleavage of caspase-3. These effects of pioglitazone were reversed by GW9662, a PPARγ antagonist, indicating that PPARγ is involved in this process. PPARγ agonists increased expression of SCD-1 that introduces a double bond on the acyl chain of long-chain fatty acid. 4-(2-Chlorophenoxy)-N-(3-(3-methylcarbamoyl)phenyl)piperidine-1-carboxamide, an inhibitor of SCD-1, abolished the anti-ER stress and anti-apoptotic effects of pioglitazone. These results suggest that PPARγ agonists attenuate palmitate-induced ER stress and apoptosis through SCD-1 induction. Up-regulation of SCD-1 may contribute to the reduction of cardiovascular events by treatment with PPARγ agonists.     

Transmembrane signaling pathway mediates oxidized low-density lipoprotein-induced expression of plasminogen activator inhibitor-1 in vascular endothelial cells

Atherosclerotic cardiovascular disease is the number one cause of death for adults in Western society. Plasminogen activator inhibitor-1 (PAI-1), the major physiological inhibitor of plasminogen activators, has been implicated in both thrombogenesis and atherogenesis. Previous studies demonstrated that copper-oxidized low-density lipoprotein (C-oLDL) stimulated production of PAI-1 in vascular endothelial cells (EC). The present study examined the involvement of lectin-like oxidized LDL receptor-1 (LOX-1) and Ras/Raf-1/ERK1/2 pathway in the upregulation of PAI-1 in cultured EC induced by oxidized LDLs. The results demonstrated that C-oLDL or FeSO(4)-oxidized LDL (F-oLDL) increased the expression of PAI-1 or LOX-1 in human umbilical vein EC (HUVEC) or coronary artery EC (HCAEC). Treatment with C-oLDL significantly increased the levels of H-Ras mRNA, protein, and the translocation of H-Ras to membrane fraction in EC. LOX-1 blocking antibody, Ras farnesylation inhibitor (FTI-277), or small interference RNA against H-Ras significantly reduced C-oLDL or LDL-induced expression of H-Ras and PAI-1 in EC. Incubation with C-oLDL or F-oLDL increased the phosphorylation of Raf-1 and ERK1/2 in EC compared with LDL or vehicle. Treatment with Raf-1 inhibitor blocked Raf-1 phosphorylation and the elevation of PAI-1 mRNA level in EC induced by C-oLDL or LDL. Treatment with PD-98059, an ERK1/2 inhibitor, blocked C-oLDL or LDL-induced ERK1/2 phosphorylation or PAI-1 expression in EC. The results suggest that LOX-1, H-Ras, and Raf-1/ERK1/2 are implicated in PAI-1 expression induced by oxidized LDLs or LDL in cultured EC.     

Correction of protein kinase C activity and macrophage migration in peripheral nerve by pioglitazone, peroxisome proliferator activated-gamma-ligand, in insulin-deficient diabetic rats

Pioglitazone, one of thiazolidinediones, a peroxisome proliferator-activated receptor (PPAR)-γ ligand, is known to have beneficial effects on macrovascular complications in diabetes, but the effect on diabetic neuropathy is not well addressed. We demonstrated the expression of PPAR-γ in Schwann cells and vascular walls in peripheral nerve and then evaluated the effect of pioglitazone treatment for 12 weeks (10 mg/kg/day, orally) on neuropathy in streptozotocin-diabetic rats. At end, pioglitazone treatment improved nerve conduction delay in diabetic rats without affecting the expression of PPAR-γ. Diabetic rats showed suppressed protein kinase C (PKC) activity of endoneurial membrane fraction with decreased expression of PKC-α. These alterations were normalized in the treated group. Enhanced expression of phosphorylated extracellular signal-regulated kinase detected in diabetic rats was inhibited by the treatment. Increased numbers of macrophages positive for ED-1 and 8-hydroxydeoxyguanosine-positive Schwann cells in diabetic rats were also corrected by the treatment. Pioglitazone lowered blood lipid levels of diabetic rats, but blood glucose and nerve sorbitol levels were not affected by the treatment. In conclusion, our study showed that pioglitazone was beneficial for experimental diabetic neuropathy via correction of impaired PKC pathway and proinflammatory process, independent of polyol pathway.     

Roscovitine inhibits ERK1/2 activation induced by angiotensin II in vascular smooth muscle cells

Roscovitine is a potent CDK inhibitor often used as a biological tool in cell-cycle studies, but its working mechanism and real targets in vascular smooth muscle cells (VSMCs) remain unclear. In this study, we observed that ERK1/2 phosphorylation induced by Ang II was abrogated by pretreating VSMCs with roscovitine for 15h. Pretreating VSMCs with roscovitine also inhibited Ang II-induced c-Jun expression and phosphorylation. We further demonstrated that roscovitine could suppress the DNA binding activity of c-Jun and activation of angiotensinogen promoter by Ang II. These results suggest that roscovitine represses Ang II-induced angiotensinogen expression by inhibiting activation of ERK1/2 and c-Jun.     

Simvastatin inhibits the additive activation of ERK1/2 and proliferation of rat vascular smooth muscle cells induced by combined mechanical stress and oxLDL through LOX-1 pathway

Vein grafts interposed into arteries are susceptible to the development of atherosclerosis due to rapid increases in blood pressure. This process is accelerated in patients with hyperlipidemia. The molecular mechanism underlying this process is unknown. In this study, quiescent rat vascular smooth muscle cells (VSMCs) were treated in vitro with mechanical stretch stress (10% elongation) with and without oxLDL (25 μg/ml) in the presence and absence of simvastatin (2.5 μmol/L). The results demonstrate that stretch stress and oxLDL can each induce activation of ERK1/2 and Ki-67 expression in VSMCs, but the peak levels of ERK activation and Ki-67 expression were observed in groups subjected to both stretch stress and oxLDL. Simvastatin was found to inhibit increased ERK activation and Ki-67 expression in VSMCs subjected to stretch stress with or without oxLDL. Mechanically, simvastatin was also found to inhibit increased expression of LOX-1 (a receptor of oxLDL) in VSMCs subjected to stretch stress with or without oxLDL. Knockdown of LOX-1 via small interfering RNAs (siRNA-LOX-1) resulted in obvious inhibition of ERK activation in VSMCs subjected to stretch stress with and without oxLDL. These results suggest that combined stretch stress and oxLDL can additively promote the activation of ERK1/2 leading to accelerated proliferation of VSMCs (e.g. increased Ki-67 expression) via LOX-1 signal pathway. This was found to be partially inhibited by simvastatin. These results may provide important data for the treatment and prevention of hypertension with or without hyperlipidemia.     

C-terminus modification of Streptomyces clavuligerus deacetoxycephalosporin C synthase improves catalysis with an expanded substrate specificity

Here we investigated the effect of pioglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, on early-phase hepatic fibrogenesis in vivo caused by acute carbon tetrachloride (CCl(4)) administration in the rat. Pioglitazone (1 mg/kg BW) prevented pericentral fibrosis and induction of alpha-smooth muscle actin (SMA) 72 h after CCl(4) administration (1 ml/kg BW). CCl(4) induction of alpha1(I)procollagen mRNA in the liver was blunted by pioglitazone to the levels almost 2/3 of CCl(4) alone. Pioglitazone also prevented CCl(4)-induced hepatic inflammation and necrosis, as well as increases in serum tumor necrosis factor-alpha levels. Further, pioglitazone inhibited the induction of alphaSMA and type I collagen in primary cultured hepatic stellate cells in a dose-dependent manner. In conclusion, pioglitazone inhibits both hepatic inflammation and activation of hepatic stellate cells, thereby ameliorating early-phase fibrogenesis in the liver following acute CCl(4).     

Activation of human peroxisome proliferator-activated receptor (PPAR) subtypes by pioglitazone

Pioglitazone, a thiazolidinedione (TZD) derivative, is an antidiabetic agent that improves hyperglycaemia and hyperlipidaemia in obese and diabetic animals via a reduction in hepatic and peripheral insulin resistance. The TZDs including pioglitazone have been identified as high affinity ligands for peroxisome proliferator-activated receptor (PPAR) gamma. The selectivity of pioglitazone for the human PPAR subtypes has not been reported, thus, we investigated the effect of pioglitazone on the human PPAR subtypes. Transient transactivation assay showed that pioglitazone is a selective hPPARgamma1 activator and a weak hPPARalpha activator. Binding assay indicated that the transactivation of hPPARgamma1 or hPPARalpha by pioglitazone is due to direct binding of pioglitazone to each subtype. Furthermore, pioglitazone significantly increased the apoA-I secretion from the human hepatoma cell line HepG2.