Mixed-lineage kinase-3 (MLK3) plays a negative modulatory role in insulin secretion from the pancreatic β-cell

Glucose-stimulated insulin secretion (GSIS) from the pancreatic β-cell in response to elevated levels of glucose is controlled by a variety of signals including intracellular calcium and nucleotides such as cAMP, ATP and GTP. These cellular signals are responsible for activation of specific kinases that mediate phosphorylation of key exocytotic proteins that lead to GSIS. In the context of protein kinases, mixed-lineage kinases (MLKs) have been implicated in an assortment of cellular functions, including cell proliferation and apoptosis. However, very little is known on potential regulatory roles of MLKs in islet β-cell function, including GSIS. The goal of this study is to determine the roles of MLK3 in GSIS. Initial data indicated that MLK3 is expressed in clonal β-cells (INS-1 832/13) and human islets. INS-1 832/13 cell exposure to stimulatory glucose (20mM) resulted in a time-dependent increase in phosphorylation of MLK3 at T277 and S281 with peak phosphorylation within 20 minutes. URMC-099, a known inhibitor of MLK3, markedly suppressed glucose-induced MLK3 phosphorylation under stimulatory glucose conditions. Moreover, URMC-099 significantly increased insulin secretion under basal (2.5mM) and stimulatory glucose exposure conditions, suggesting a suppressive role for MLK3 in insulin secretion. Together, these studies provide evidence that MLK3 is in fact expressed in clonal and primary β-cells and inhibition of glucose-induced phosphorylation of MLK3 results in potentiation of GSIS. Studies are underway to further confirm our pharmacological findings using molecular biological (siRNA-MLK3) approaches

In vitro and in vivo effect of carmustine and selenite combination on EGFR signaling in androgen-independent prostate cancer

Introduction: Despite the use of androgen deprivation therapy, the majority of prostate cancer patients will progress to castration resistant disease within 2-3 years, driven by aberrant androgen receptor activation. We have previously shown that combination of carmustine and selenite effectively induces apoptosis by completely reducing AR and AR-variants in AR-dependent castration resistant prostate cancer cells both in vitro and in vivo. Although studies have demonstrated that AR signaling is a central mechanism of castration resistant prostate cancer progression, the cell growth mediated by AR-independent signaling is also operative. EGFR signaling has been implicated in the survival, invasion, and metastasis of prostate cancer cells in an AR-independent manner. Therefore, in this study, we tested whether the combination of carmustine and selenite could inhibit EGFR signaling, induce apoptosis, and inhibit growth of AR-independent prostate cancer cells in an in vitro cell culture and in vivo Methods: AR negative PC-3 prostate cancer cell line was used to study the effect of carmustine and selenite alone or in combination on EGFR signaling. Cell viability, proliferation, and apoptosis in the presence or absence of EGF (50ng/ml) were determined. Dose and time dependent effect of EGF on the activation of EGFR was studied. The carmustine and/or selenite effect on EGF stimulated phospho-EGFR, and its downstream signaling pathways, such as AKT and MAPK, were studied in comparison with inhibitors of EGFR (AG1478) or PI3 kinase (LY294002) or MAPK (PD98059). The in vivo effect of carmustine and selenite was examined on PC-3 tumor growth in athymic nude mice. Results: Combination of carmustine and selenite treatment in the presence of EGF markedly suppressed cell viability and proliferation of PC-3 cells. EGF exposure increased phospho-EGFR (Tyr845, Tyr992, Tyr1068, and Tyr1045), pAkt (Ser473), and pERK1/2 (Thr204/Tyr202) protein expression levels in PC-3 cells in a dose and time dependent manner. The combination treatment exhibited a strong inhibitory effect on EGF stimulated EGFR, Akt, and ERK1/2 phosphorylation. Combination treatment was able to induce apoptosis even in the presence of EGF. Consistent with in vitro results, combination treatment for 3 weeks reduced PC-3 xenograft tumors in nude mice by 88% without any toxicity. Whereas individual agent treatment showed only partial effect both in vitro and in vivo. Conclusions: Our pre-clinical data demonstrate that the combination treatment of carmustine and selenite substantially inhibits EGFR signaling, induces apoptosis, and reduces tumor growth of AR-independent prostate cancer cells in vitro and in vivo. Our novel findings suggest that the combination of carmustine and selenite is an effective therapeutic agent for successful treatment, survival, and improved quality of life for patients with prostate cancer

The role of the cytoskeleton in differentially regulating pressure-mediated effects on malignant colonocyte focal adhesion signaling and cell adhesion. Carcinogenesis 26

Increased extracellular pressure stimulates colon cancer cell adhesion by activating focal adhesion kinase (FAK) and Src. We investigated the role of the cytoskeleton in pressure-induced inside-out FAK and Src phosphorylation and pressure-stimulated adhesion. We perturbed actin polymerization with phalloidin, cytochalasin D and latrunculin B, and microtubule organization with colchicine and paclitaxol. We compared the effects of these agents on pressure-induced SW620 and human primary colon cancer cell adhesion and inside-out FAK/Src activation with outside-in adhesion-dependent FAK/Src activation. Cells pretreated with cytoskeletal inhibitors were subjected to 15 mmHg increased pressure and allowed to adhere to collagen I coated plates or prevented from adhesion to pacificated plates for 30 min. Phalloidin, cytochalasin D, latrunculin B and colchicine pretreatment completely prevented pressure-stimulated and significantly inhibited basal SW620 cell adhesion. Taxol did not inhibit pressure-induced colon cancer cell adhesion, but significantly lowered basal adhesion. Cytochalasin D and colchicine had similar effects in pressure-stimulated primary human malignant colonocytes. Phalloidin, cytochalasin D, latrunculin B and colchicine prevented pressure-induced SW620 FAK phosphorylation but not Src phosphorylation. FAK phosphorylation in response to collagen I adhesion was significantly attenuated but not completely prevented by these inhibitors. Although Src phosphorylation was not increased on adhesion, the cytoskeleton disrupting agents significantly lowered basal Src phosphorylation in adherent cells. These results suggest that both cytoskeleton-dependent FAK activation and cytoskeletonindependent Src activation may be required for extracellular pressure to stimulate colon cancer cell adhesion. Furthermore, the cytoskeleton plays a different role in pressure-activated FAK and Src signaling than in FAK and Src activation in adherent cells. We, therefore, hypothesize that cytoskeletal interactions with focal adhesion signals mediate the effects of extracellular pressure on colon cancer cell adhesion

Smad2/3 Activation Regulates Smad1/5/8 Signaling via a Negative Feedback Loop to Inhibit 3T3-L1 Adipogenesis

Adipose tissues (AT) expand in response to energy surplus through adipocyte hypertrophy and hyperplasia. The latter, also known as adipogenesis, is a process by which multipotent precursors differentiate to form mature adipocytes. This process is directed by developmental cues that include members of the TGF-β family. Our goal here was to elucidate, using the 3T3-L1 adipogenesis model, how TGF-β family growth factors and inhibitors regulate adipocyte development. We show that ligands of the Activin and TGF-β families, several ligand traps, and the SMAD1/5/8 signaling inhibitor LDN-193189 profoundly suppressed 3T3-L1 adipogenesis. Strikingly, anti-adipogenic traps and ligands engaged the same mechanism of action involving the simultaneous activation of SMAD2/3 and inhibition of SMAD1/5/8 signaling. This effect was rescued by the SMAD2/3 signaling inhibitor SB-431542. By contrast, although LDN-193189 also suppressed SMAD1/5/8 signaling and adipogenesis, its effect could not be rescued by SB-431542. Collectively, these findings reveal the fundamental role of SMAD1/5/8 for 3T3-L1 adipogenesis, and potentially identify a negative feedback loop that links SMAD2/3 activation with SMAD1/5/8 inhibition in adipogenic precursors

Oxalate-induced activation of PKC-α and -δ regulates NADPH oxidase-mediated oxidative injury in renal tubular epithelial cells

Oxalate-induced oxidative stress contributes to cell injury and promotes renal deposition of calcium oxalate crystals. However, we do not know how oxalate stimulates reactive oxygen species (ROS) in renal tubular epithelial cells. We investigated the signaling mechanism of oxalate-induced ROS formation in these cells and found that oxalate significantly increased membrane-associated protein kinase C (PKC) activity while at the same time lowering cytosolic PKC activity. Oxalate markedly translocated PKC-α and -δ from the cytosol to the cell membrane. Pretreatment of LLC-PK1 cells with specific inhibitors of PKC-α or -δ significantly blocked oxalate-induced generation of superoxide and hydrogen peroxide along with NADPH oxidase activity, LDH release, lipid hydroperoxide formation, and apoptosis. The PKC activator PMA mimicked oxalate's effect on oxidative stress in LLC-PK1 cells as well as cytosol-to-membrane translocation of PKC-α and -δ. Silencing of PKC-α expression by PKC-α-specific small interfering RNA significantly attenuated oxalate-induced cell injury by decreasing hydrogen peroxide generation and LDH release. We believe this is the first demonstration that PKC-α- and -δ-dependent activation of NADPH oxidase is one of the mechanisms responsible for oxalate-induced oxidative injury in renal tubular epithelial cells. The study suggests that the therapeutic approach might be considered toward attenuating oxalate-induced PKC signaling-mediated oxidative injury in recurrent stone formers