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Human bone marrow-derived mesenchymal stem cells (MSCs) have the unique ability to home toward injuries or tumor sites. We have previously shown that the tumor-tropic property is dependent on the intrinsic expression and activity of the matrix remodeling gene, matrix metalloproteinase 1 (MMP-1). Herein, crosstalk between MMP-1/protease activated receptor 1 (PAR-1) and the G-protein coupled receptor stromal-derived growth factor 1 (SDF-1)/C-X-C chemokine receptor 4 (CXCR-4) in facilitating cell migration was investigated. Gain-of-function and RNA interference (RNAi) technology were used to evaluate the interplay between the key players. The downstream effect on the tumor-tropic migration of MSCs was investigated using modified Boyden chamber assay. Neutralizing PAR-1 activation using monoclonal antibody and targeted knockdown of MMP-1 using RNAi resulted in decreased expression of SDF-1, which was not observed in control-RNAi-transfected cells. Over-expression of CXCR-4 failed to promote MSC migration; the percentage of migrated cells toward tumor cell conditioned medium was similar to the vector-transduced and the CXCR-4-transduced MSCs. Furthermore, inhibition of SDF-l/CXCR-4 signaling using AMD3100 reduced MSC migration through the deregulation of MMP-1 promoter activities, protein expression, and metalloproteinase activity. Collectively, our results showed that MMP-l-mediated MSC tumor tropism is dependent on crosstalk with the SDF-1/CXCR-4 axis. [ABSTRACT FROM AUTHOR]

Aberrant β-adrenergic signaling and depressed calcium homeostasis, associated with an imbalance of protein kinase A and phosphatase-1 activities, are hallmarks of heart failure. Phosphatase-1 is restrained by its endogenous inhibitor, protein phosphatase inhibitor-1 (PPI-1). We assessed 352 normal subjects, along with 959 patients with heart failure and identified a polýanorphism in PPI-1 (G147D) exclusively in black subjects. To determine whether the G147D variant could affect cardiac function, we infected adult cardiomyocytes with adenoviruses expressing D147 or wild-type (G147) PPI-1. Under basal con- ditious, there were no significant differences in fractional shortening or contraction or relaxation rates. However, the enhancement of contractile parameters after isoproterenol stimulation was significantly blunted in D147 compared with G147 and control myocytes. Similar findings were observed in calcium kinetics. The attenuated β-agonist response was associated with decreased (50%) phosphorylation of phos- pholamban (PLN) at serine 16, whereas phosphorylation of troponin I and ryanodine receptor was unaltered. These findings suggest that the human G147D PPI-1 can attenuate responses of cardiomyocytes to β-adrenergic agonists by decreasing PLN phosphorylation and therefore may contribute to deteriorated function in heart failure. [ABSTRACT FROM AUTHOR]

Activation of the β-adrenergic; receptor elicits phosphorylation of phospholamban, leading to enhanced cardiac contractility. Reversal of this process is mediated by protein phosphatase 1, which is under regulation by endogenous Inhibitor-1 (I-1). We have identified a polymorphism in human I-1, entailing substitution of glycine at AA147 with aspartic acid (G147D) in 29/288 black patients of heart failure and none of 671 whites. To clarify the functional significance of the GI47D I-1, we infected isolated adult rat cardiomyocytes with an adenovirus expressing G147D I-1, wild type I-1, or control GFP. Under basal conditions, there were no significant differences in fractional shortening, contraction rate or relaxation rate among the three groups. However, the responses of these parameters to isoproterenol were significantly blunted in cardiomyocytes infected with the G147D variant, compared to the other two groups. Consistent with these mechanical parameters, the velocity of Ca transient decay was significantly delayed and phosphorylation of phospholamban was decreased by 50% in G147D-cells. These findings suggest that the human G147D 1-1 polymorphism attenuates the heart's responses to β-adrenergic agonists through decreased PLN phosphorylation, which may contribute to the deteriorated function of the failing heart. [ABSTRACT FROM AUTHOR]

The secreted form of SP-D is a large multimer formed by assembly of trimeric and dodecameric subunits. Higher oligomerization of SP-D is dependent upon 2 cysteine residues within its tail domain. In vitro treatment of recombinant SP-D with S-nitrocysteine (SNOC) results in formation of a S-nitrosylated isoform (SNO-SP-D). Structurally, SNO modification disrupted SP-D oligomerization into dodecamers, trimers, and monomers; functionally SNO-SP-D markedly enhanced chemotaxis and p38 phosphorylation in RAW 264 macrophages. Previously we have shown in several models of lung inflammation, including bleomycin injury, that iNOS mRNA and total BAL NO content are increased. Native electrophoresis of BAL from bleomycin treated mice revealed that lung injury induced S-nitrosylation and disrupted SP-D oligomerization. The chemotactic effect on macrophages by bleomycin BAL was inhibited by anti-SP-D. We conclude that SP-D is S-nitrosylated by NO in vitro and in vivo and that this modification is associated with alterations in higher order structure resulting in a functional switch from inflammatory inhibitor to activator. [ABSTRACT FROM AUTHOR]