Protein kinase C-ß-dependent changes in the glucose metabolism of bone marrow stromal cells of chronic lymphocytic leukemia.

Survival of chronic lymphocytic leukemia (CLL) cells critically depends on the support of an adapted and therefore appropriate tumor microenvironment. Increasing evidence suggests that B-cell receptor-associated kinases such as protein kinase C-ß (PKCß) or Lyn kinase are essential for the formation of a microenvironment supporting leukemic growth. Here, we describe the impact of PKCß on the glucose metabolism in bone marrow stromal cells (BMSC) upon CLL contact. BMSC get activated by CLL contact expressing stromal PKCß that diminishes mitochondrial stress and apoptosis in CLL cells by stimulating glucose uptake. In BMSC, the upregulation of PKCß results in increased mitochondrial depolarization and leads to a metabolic switch toward oxidative phosphorylation. In addition, PKCß-deficient BMSC regulates the expression of Hnf1 promoting stromal insulin signaling after CLL contact. Our data suggest that targeting PKCß and the glucose metabolism of the leukemic niche could be a potential therapeutic strategy to overcome stroma-mediated drug resistance.

Remifentanil attenuates lipopolysaccharide-induced oxidative injury by downregulating PKCß2 activation and inhibiting autophagy in H9C2 cardiomyocytes.

AIM: Lipopolysaccharide (LPS)-induced myocardial injury is a leading cause of death in patients with sepsis, which is associated with excessive activation of PKCß (especially PKCß2) and autophagy. Remifentanil, a µ-opioid receptor agonist, is well demonstrated to have beneficial effects during sepsis, but the underlying mechanisms are still unknown. The present study was designed to investigate the roles of remifentanil in PKCß2 and autophagy in LPS-treated cardiomyocytes. MAIN METHODS: H9C2 cardiomyocytes were treated with or without remifentanil (2.5 µM), PKCß2 inhibitor CGP53353 (CGP, 1 µM) or autophagy inhibitor 3-methyladenine (3-MA, 10 µM) in the presence or absence of LPS (10 µg/mL). KEY FINDINGS: LPS exposure for 24 h led to a significant increase in cell death, LDH release and MDA production in H9C2 cardiomyocytes, accompanied with decreased SOD activity and excessive PKCß2 activation and autophagy indicated by enhanced Beclin-1 and LC-3II expression and decreased p62 expression. All these changes were attenuated by remifentanil intervention. In addition, inhibition of LPS-induced PKCß2 activation by CGP or autophagy inhibitor 3-MA has similar effects to remifentanil. SIGNIFICANCE: Remifentanil protects H9C2 cardiomyocytes against LPS-induced oxidative injury, as a result of downregulating PKCß2 activation and inhibiting autophagy, partially.

Identification of protein kinase C activation as a novel mechanism for RGS2 protein upregulation through phenotypic screening of natural product extracts.

Biochemical high-throughput screening is widely used in drug discovery, using a variety of small molecule libraries. However, broader screening strategies may be more beneficial to identify novel biologic mechanisms. In the current study we used a ß-galactosidase complementation method to screen a selection of microbial-derived pre-fractionated natural product extracts for those that increase regulator of G protein signaling 2 (RGS2) protein levels. RGS2 is a member of a large family of proteins that all regulate signaling through G protein-coupled receptors (GPCRs) by accelerating GTPase activity on active Gα as well as through other mechanisms. RGS2(-/-) mice are hypertensive, show increased anxiety, and are prone to heart failure. RGS2 has a very short protein half-life due to rapid proteasomal degradation, and we propose that enhancement of RGS2 protein levels could be a beneficial therapeutic strategy. Bioassay-guided fractionation of one of the hit strains yielded a pure compound, Indolactam V, a known protein kinase C (PKC) activator, which selectively increased RGS2 protein levels in a time- and concentration-dependent manner. Similar results were obtained with phorbol 12-myristate 13-acetate as well as activation of the Gq-coupled muscarinic M3 receptor. The effect on RGS2 protein levels was blocked by the nonselective PKC inhibitor Gö6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), the PKCß-selective inhibitor Ruboxastaurin, as well as small interfering RNA-mediated knockdown of PKCß. Indolactam V-mediated increases in RGS2 protein levels also had functional effects on GPCR signaling. This study provides important proof-of-concept for our screening strategy and could define a negative feedback mechanism in Gq/Phospholipase C signaling through RGS2 protein upregulation.