Rap1b in smooth muscle and endothelium is required for maintenance of vascular tone and normal blood pressure.

OBJECTIVE: Small GTPase Ras-related protein 1 (Rap1b) controls several basic cellular phenomena, and its deletion in mice leads to several cardiovascular defects, including impaired adhesion of blood cells and defective angiogenesis. We found that Rap1b(-/-) mice develop cardiac hypertrophy and hypertension. Therefore, we examined the function of Rap1b in regulation of blood pressure. APPROACH AND RESULTS: Rap1b(-/-) mice developed cardiac hypertrophy and elevated blood pressure, but maintained a normal heart rate. Correcting elevated blood pressure with losartan, an angiotensin II type 1 receptor antagonist, alleviated cardiac hypertrophy in Rap1b(-/-) mice, suggesting a possibility that cardiac hypertrophy develops secondary to hypertension. The indices of renal function and plasma renin activity were normal in Rap1b(-/-) mice. Ex vivo, we examined whether the effect of Rap1b deletion on smooth muscle-mediated vessel contraction and endothelium-dependent vessel dilation, 2 major mechanisms controlling basal vascular tone, was the basis for the hypertension. We found increased contractility on stimulation with a thromboxane analog or angiotensin II or phenylephrine along with increased inhibitory phosphorylation of myosin phosphatase under basal conditions consistent with elevated basal tone and the observed hypertension. Cyclic adenosine monophosphate-dependent relaxation in response to Rap1 activator, Epac, was decreased in vessels from Rap1b(-/-) mice. Defective endothelial release of dilatory nitric oxide in response to elevated blood flow leads to hypertension. We found that nitric oxide-dependent vasodilation was significantly inhibited in Rap1b-deficient vessels. CONCLUSIONS: This is the first report to indicate that Rap1b in both smooth muscle and endothelium plays a key role in maintaining blood pressure by controlling normal vascular tone.

The cAMP-responsive Rap1 guanine nucleotide exchange factor, Epac, induces smooth muscle relaxation by down-regulation of RhoA activity.

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.

Lithocholic Acid Hydroxyamide Destabilizes Cyclin D1 and Induces G0/G1 Arrest by Inhibiting Deubiquitinase USP2a.

USP2a is a deubiquitinase responsible for stabilization of cyclin D1, a crucial regulator of cell-cycle progression and a proto-oncoprotein overexpressed in numerous cancer types. Here we report that lithocholic acid (LCA) derivatives are inhibitors of USP proteins, including USP2a. The most potent LCA derivative, LCA hydroxyamide (LCAHA), inhibits USP2a, leading to a significant Akt/GSK3ß-independent destabilization of cyclin D1, but does not change the expression of p27. This leads to the defects in cell-cycle progression. As a result, LCAHA inhibits the growth of cyclin D1-expressing, but not cyclin D1-negative cells, independently of the p53 status. We show that LCA derivatives may be considered as future therapeutics for the treatment of cyclin D1-addicted p53-expressing and p53-defective cancer types.

Structural basis for small molecule targeting of the programmed death ligand 1 (PD-L1).

Targeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has provided unprecedented results in cancer treatment in the recent years. Development of chemical inhibitors for this pathway lags the antibody development because of insufficient structural information. The first nonpeptidic chemical inhibitors that target the PD-1/PD-L1 interaction have only been recently disclosed by Bristol-Myers Squibb. Here, we show that these small-molecule compounds bind directly to PD-L1 and that they potently block PD-1 binding. Structural studies reveal a dimeric protein complex with a single small molecule which stabilizes the dimer thus occluding the PD-1 interaction surface of PD-L1s. The small-molecule interaction "hot spots" on PD-L1 surfaces suggest approaches for the PD-1/PD-L1 antagonist drug discovery.

Fluorescence linked enzyme chemoproteomic strategy for discovery of a potent and selective DAPK1 and ZIPK inhibitor.

DAPK1 and ZIPK (also called DAPK3) are closely related serine/threonine protein kinases that regulate programmed cell death and phosphorylation of non-muscle and smooth muscle myosin. We have developed a fluorescence linked enzyme chemoproteomic strategy (FLECS) for the rapid identification of inhibitors for any element of the purinome and identified a selective pyrazolo[3,4-d]pyrimidinone (HS38) that inhibits DAPK1 and ZIPK in an ATP-competitive manner at nanomolar concentrations. In cellular studies, HS38 decreased RLC20 phosphorylation. In ex vivo studies, HS38 decreased contractile force generated in mouse aorta, rabbit ileum, and calyculin A stimulated arterial muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor also promoted relaxation in Ca(2+)-sensitized vessels. A close structural analogue (HS43) with 5-fold lower affinity for ZIPK produced no effect on cells or tissues. These findings are consistent with a mechanism of action wherein HS38 specifically targets ZIPK in smooth muscle. The discovery of HS38 provides a lead scaffold for the development of therapeutic agents for smooth muscle related disorders and a chemical means to probe the function of DAPK1 and ZIPK across species.

The actin associated protein palladin is important for the early smooth muscle cell differentiation.

Palladin, an actin associated protein, plays a significant role in regulating cell adhesion and cell motility. Palladin is important for development, as knockdown in mice is embryonic lethal, yet its role in the development of the vasculature is unknown. We have shown that palladin is essential for the expression of smooth muscle cells (SMC) marker genes and force development in response to agonist stimulation in palladin deficient SMCs. The goal of the study was to determine the molecular mechanisms underlying palladin's ability to regulate the expression of SMC marker genes. Results showed that palladin expression was rapidly induced in an A404 cell line upon retinoic acid (RA) induced differentiation. Suppression of palladin expression with siRNAs inhibited the expression of RA induced SMC differentiation genes, SM α-actin (SMA) and SM22, whereas over-expression of palladin induced SMC gene expression. Chromatin immunoprecipitation assays provided evidence that palladin bound to SMC genes, whereas co-immunoprecipitation assays also showed binding of palladin to myocardin related transcription factors (MRTFs). Endogenous palladin was imaged in the nucleus, increased with leptomycin treatment and the carboxyl-termini of palladin co-localized with MRTFs in the nucleus. Results support a model wherein palladin contributes to SMC differentiation through regulation of CArG-SRF-MRTF dependent transcription of SMC marker genes and as previously published, also through actin dynamics. Finally, in E11.5 palladin null mouse embryos, the expression of SMA and SM22 mRNA and protein is decreased in the vessel wall. Taken together, our findings suggest that palladin plays a key role in the differentiation of SMCs in the developing vasculature.