Stabilization of the prostate‐specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim‐1 in prostate cancer cells

Loss of NKX3.1 is an early and consistent event in prostate cancer and is associated with increased proliferation of prostate epithelial cells and poor prognosis. NKX3.1 stability is regulated post‐translationally through phosphorylation at multiple sites by several protein kinases. Here, we report the paradoxical stabilization of the prostate‐specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim‐1 in prostate cancer cells. Pharmacologic Pim‐1 inhibition using the small molecule inhibitor CX‐6258 decreased steady state levels and half‐life of NKX3.1 protein but mRNA was not affected. This effect was reversed by inhibition of the 26S‐proteasome, demonstrating that Pim‐1 protects NKX3.1 from proteasome‐mediated degradation. Mass spectrometric analyses revealed Thr89, Ser185, Ser186, Ser195, and Ser196 as Pim‐1 phospho‐acceptor sites on NKX3.1. Through mutational analysis, we determined that NKX3.1 phosphorylation at Ser185, Ser186, and within the N‐terminal PEST domain is essential for Pim‐1‐mediated stabilization. Further, we also identified Lys182 as a critical residue for NKX3.1 stabilization by Pim‐1. Pim‐1‐mediated NKX3.1 stabilization may be important in maintaining normal cellular homeostasis in normal prostate epithelial cells, and may maintain basal NKX3.1 protein levels in prostate cancer cells. J. Cell. Biochem. 114: 1050–1057, 2013. © 2012 Wiley Periodicals, Inc.     

Fasudil hydrochloride hydrate, a Rho-kinase inhibitor, suppresses isoproterenol-induced heart failure in rats via JNK and ERK1/2 pathways

The Rho-kinase (ROCK) plays an important role in the pathogenesis of heart injury. Recent cellular and molecular biology studies indicated a pivotal role of the RhoA/ROCK cascade in many aspects of cardiovascular function such as heart failure, cardiac hypertrophy, and ventricular remodeling following myocardial infarction. However, the signal transduction of RhoA/ROCK and its down-stream signaling pathways remains elusive, and the mechanism of ROCK-mediated isoproterenol (ISO)-induced heart failure is still not thoroughly understood. In the present study, we investigated the effect of the ROCK inhibitor, fasudil hydrochloride hydrate, on ISO-induced heart failure and the potential relationship of RhoA/ROCK to the extracellular signal-regulated kinases (ERK) and the c-jun NH 2-terminal kinase (JNK) pathways. Male Sprague-Dawley (SD) rats, maintained on a normal diet, were randomly divided into four groups given control, ISO alone, ISO with low-dose fasudil, or ISO with high-dose fasudil treatments. Fasudil effectively inhibited ISO-induced heart failure, as evaluated by biometric, hemodynamic, and histological examinations. Consistently, ISO-induced ROCK-1 mRNA expression and myosin phosphatase target subunit-1 (MYPT-1) phosphorylation were markedly suppressed by fasudil. In addition, fasudil significantly decreased ISO-induced JNK activation, ERK translocation to the nucleus and subsequent c-fos, c-jun expression and upregulated c-FLIP(L) expression. Taken together, these results indicate that the RhoA/ROCK pathway is essential for ISO induced heart failure, which can be effectively suppressed by fasudil.     

CaM kinase control of AKT and LNCaP cell survival

AKT and its substrate BAD have been shown to promote prostate cancer cell survival. Agonists, such as carbachol, and hormones that increase intracellular calcium concentration can activate AKT leading to cancer cell survival. The LNCaP prostate cancer cells express the carbachol-sensitive M(3) -subtype of G protein-coupled receptors that cause increases in intracellular calcium and activate the family of Ca(2+) /calmodulin-dependent protein kinases (CaM Ks). One type of CaM Kinase, CaM Kinase Kinase (CaM KK), phosphorylates several substrates including AKT on threonine 308. AKT phosphorylation and activation enhances cell survival through phosphorylation of BAD protein and the subsequent blockade of caspase activation. Our goals were to examine the mechanism of carbachol activation of AKT and BAD in LNCaP prostate cancer cells and evaluate whether CaM KK may be mediating carbachol's activation of AKT and cell survival. Our results suggest that carbachol treatment of LNCaP cells promoted cell survival through CaM KK and its phosphorylation of AKT. The bacterial toxin anisomycin triggered caspase-3 activation in LNCaP cells that was blocked by carbachol in a CaM KK- and AKT-dependent manner. AKT and BAD phosphorylation were blocked by the selective CaM KK inhibitor, STO-609, as well as siRNA directed against CaM KK. BAD phosphorylation was also blocked by treating cells with the AKT inhibitor, AKT-X, as well as siRNA to AKT. Additionally, epinephrine promoted LNCaP cell survival through activation of AKT that was insensitive to STO-609. Taken together these data suggest a survival role for CaM KK operating through AKT and BAD in LNCaP prostate cancer cells.     

Ganglioside GM3 inhibits hepatoma cell motility via down‐regulating activity of EGFR and PI3K/AKT signaling pathway

Two related sublines derived from murine ascites hepatoma cell lines Hca‐F25, which were selected for their markedly different metastatic potential to lymph nodes, were found to be distinct in their ganglioside patterns. The low metastatic cell line (HcaP) contained a major ganglioside GM3, whereas the high metastatic cell line (HcaF) contained a major ganglioside GM2. Suppression of GM3 by P4 enhanced the mobility and migration of the low metastatic HcaP cells in vitro. Increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 inhibited the mobility and migration. These results suggested that the differences in lymphatic metastasis potential between these two cell lines could be attributed to the differences in their ganglioside compositions, and GM3 could suppress the motility and migration of these cells. Further, we investigated the mechanism by which GM3 suppressed the cell mobility and migration. The results showed that suppression of GM3 synthesis by P4 in low metastatic HcaP cells promoted PKB/Akt phosphorylation at Ser473 and Thr308, and phosphorylation of EGFR at the Tyr1173. In contrast, increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 into the culture medium suppressed phosphorylation of PKB/Akt and EGFR at the same residues. Taken together, these results suggested that the mechanism of GM3‐suppressed cell motility and migration may involve the inhibition of phosphorylation of EGFR and the activity of PI3K/AKT signaling pathway. J. Cell. Biochem. 114: 1616–1624, 2013. © 2013 Wiley Periodicals, Inc.     

Suppression of the PI3K subunit p85α delays embryoid body development and inhibits cell adhesion

Phosphatidylinositol-3-kinases (PI3Ks) exert a variety of signaling functions in eukaryotes. We suppressed the PI3K regulatory subunit p85α using a small interfering RNA (Pik3r1 siRNA) and examined the effects on embryoid body (EB) development in hanging drop culture. We observed a 150% increase in the volume of the treated EBs within 24 h, compared to the negative controls. Fluorescence Activated Cell Sorting (FACS) assays showed that this increase in volume is not due to increased cellular proliferation. Instead, the increase in volume appears to be due to reduced cellular aggregation and adherence. This is further shown by our observation that 40% of treated EBs form twin instead of single EBs, and that they have a significantly reduced ability to adhere to culture dishes when plated. A time course over the first 96 h reveals that the impaired adherence is transient and explained by an initial 12-hour delay in EB development. Quantitative PCR expression analysis suggests that the adhesion molecule integrin-β1 (ITGB1) is transiently downregulated by the p85α suppression. In conclusion we found that suppressing p85α leads to a delay in forming compact EBs, accompanied by a transient inability of the EBs to undergo normal cell-cell and cell-substrate adhesion.     

Nestin protects mouse podocytes against high glucose-induced apoptosis by a Cdk5-dependent mechanism

Podocyte apoptosis contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms that mediate hyperglycemia‐induced podocyte apoptosis remain poorly understood. Recent findings indicate that the disruption of the cytoskeleton is related to the podocyte apoptosis. In the present study, we investigated the involvement of nestin, an important cytoskeleton‐associated class VI intermediate filament (IF) protein, in the high glucose (HG)‐induced podocyte apoptosis. Our data showed that HG decreased the expression level of nestin, either mRNA or protein, in a time‐dependent manner in cultured podocytes. Also, through knockdown of nestin expression by miRNA interference, the HG‐induced podocyte apoptotic rate was significantly increased. The expression of cleaved caspase‐3 was also markedly elevated. Considering that nestin is a substrate of cyclin‐dependent kinase 5 (Cdk5), we further assessed the expression of Cdk5 in HG‐treated podocytes. The results showed that HG stimulation increased the protein and mRNA expression of Cdk5 in a time‐dependent manner in cultured mouse podocytes. The protein activator of Cdk5, p35, was also increased in a time‐dependent manner by HG stimulation, and downregulation of Cdk5 by miRNA interference attenuated the nestin reduction in HG‐treated podocytes; the HG‐induced podocyte apoptosis, the increased cleaved caspase‐3 expression and the Bax/Bcl‐2 ratio were all effectively attenuated. These data suggested that nestin, which is dependent on Cdk5 regulation, plays a cytoprotective role in HG‐induced podocyte apoptosis. J. Cell. Biochem. 113: 3186–3196, 2012. © 2012 Wiley Periodicals, Inc.