Cadherin-7 interacts with melanoma inhibitory activity protein and negatively modulates melanoma cell migration.

Melanoma inhibitory activity (MIA) has been identified as a small protein secreted from malignant melanoma cells, which strongly enhances melanoma cell migration and invasion. Detailed analyses performed by our group showed interaction of MIA with extracellular matrix proteins and integrin alpha4beta1 and alpha5beta1 leading to cellular detachment. In this study, we identified cadherin-7 as a new MIA-binding protein using surface-enhanced laser desorption/ionization-mass spectrometry technology and co-immunoprecipitation. Cadherin-7 is a classical cell-cell adhesion molecule which was shown to be upregulated in malignant melanoma. We demonstrated enhanced expression of cadherin-7 in primary tumor cells compared to metastatic cells. Upregulation of cadherin-7 expression in metastatic cell lines but also downregulation of expression in cells derived from primary melanomas resulted in reduced cell migration. In addition, we speculate that MIA/cadherin-7 interaction may regulate cell-cell adhesion of malignant melanoma cells influencing the migration of the cells. Interestingly, overexpression of cadherin-7 resulted in a decreased MIA mRNA expression. In addition, MIA effects on cell migration were abrogated in cell clones overexpressing cadherin-7. In conclusion, these findings suggest that cadherin-7 regulates the expression and activity of MIA and the migration of melanoma cells playing a role in tumor development of malignant melanoma.

Fat accumulation with altered inflammation and regeneration in skeletal muscle of CCR2-/- mice following ischemic injury.

Chemokines recruit inflammatory cells to sites of injury, but the role of the CC chemokine receptor 2 (CCR2) during regenerative processes following ischemia is poorly understood. We studied injury, inflammation, perfusion, capillary formation, monocyte chemotactic protein-1 (MCP-1) levels, muscle regeneration, fat accumulation, and transcription factor activation in hindlimb muscles of CCR2-/- and wild-type (WT) mice following femoral artery excision (FAE). In both groups, muscle injury and restoration of vascular perfusion were similar. Nevertheless, edema and neutrophil accumulation were significantly elevated in CCR2-/- compared with WT mice at day 1 post-FAE and fewer macrophages were present at day 3. MCP-1 levels in post-ischemic calf muscle of CCR2-/- animals were significantly elevated over baseline through 14 days post-FAE and were higher than WT mice at days 1, 7, and 14. In addition, CCR2-/- mice exhibited impaired muscle regeneration, decreased muscle fiber size, and increased intermuscular adipocytes with similar capillaries/mm(2) postinjury. Finally, the transcription factors, MyoD and signal transducers of and activators of transcription-3 (STAT3), were significantly increased above baseline but did not differ significantly between groups at any time point post-FAE. These findings suggest that increases in MCP-1, and possibly, MyoD and STAT3, may modulate molecular signaling in CCR2-/- mice during inflammatory and regenerative events. Furthermore, alterations in neutrophil and macrophage recruitment in CCR2-/- mice may critically alter the normal progression of downstream regenerative events in injured skeletal muscle and may direct myogenic precursor cells in the regenerating milieu toward an adipogenic phenotype.

MCP-1 deficiency causes altered inflammation with impaired skeletal muscle regeneration.

We examined the role of MCP-1, a potent chemotactic and activating factor for macrophages, in perfusion, inflammation, and skeletal muscle regeneration post-ischemic injury. MCP-1-/- or C57Bl/6J control mice [wild-type (WT)] underwent femoral artery excision (FAE). Muscles were collected for histology, assessment of tissue chemokines, and activity measurements of lactate dehydrogenase (LDH) and myeloperoxidase. In MCP-1-/- mice, restoration of perfusion was delayed, and LDH and fiber size, indicators of muscle regeneration, were decreased. Altered inflammation was observed with increased neutrophil accumulation in MCP-1-/- versus WT mice at Days 1 and 3 (P< or =0.003), whereas fewer macrophages were present in MCP-1-/- mice at Day 3. As necrotic tissue was removed in WT mice, macrophages decreased (Day 7). In contrast, macrophage accumulation in MCP-1-/- was increased in association with residual necrotic tissue and impaired muscle regeneration. Consistent with altered inflammation, neutrophil chemotactic factors (keratinocyte-derived chemokine and macrophage inflammatory protein-2) were increased at Day 1 post-FAE. The macrophage chemotactic factor MCP-5 was increased significantly in WT mice at Day 3 compared with MCP-1-/- mice. However, at post-FAE Day 7, MCP-5 was significantly elevated in MCP-1-/- mice versus WT mice. Addition of exogenous MCP-1 did not induce proliferation in murine myoblasts (C2C12 cells) in vitro. MCP-1 is essential for reperfusion and the successful completion of normal skeletal muscle regeneration after ischemic tissue injury. Impaired muscle regeneration in MCP-1-/- mice suggests an important role for macrophages and MCP-1 in tissue reparative processes.

MCP-1 parallels inflammatory and regenerative responses in ischemic muscle.

BACKGROUND: Monocyte chemotactic protein-1 (MCP-1) is important in macrophage recruitment and activation. However, the magnitude and temporal sequence of MCP-1 expression in relation to tissue injury and regeneration following ischemic injury remains unknown. MATERIALS AND METHODS: Hind limb ischemia was induced by femoral artery excision (FAE) in C57Bl/6J mice; a sham surgery was performed on the contralateral leg. Muscle lysates were used to measure MCP-1 and activities of creatine kinase, lactate dehydrogenase, and myeloperoxidase. Histology and immunohistochemistry were used to localize inflammation and MCP-1. RESULTS: FAE resulted in a prolonged period of ischemia and the administration of MCP-1 did not alter the restoration of perfusion. One day after femoral artery excision, extensive muscle necrosis and neutrophils were prevalent throughout the musculature of the lower leg. By 3 days, a mononuclear cell infiltrate predominated in association with robust muscle regeneration as indicated by myoD expression. Concomitantly, myeloperoxidase was maximally increased. Muscle enzymes (creatine kinase and lactate dehydrogenase) were maximally decreased within 3 days and returned to baseline levels by day 14, a time course consistent with injury and regeneration observed by histology. In parallel with these inflammatory and regenerative events, MCP-1 in muscle was maximally increased at day 3. By immunohistochemistry, MCP-1 was within vascular endothelial cells and infiltrating macrophages in areas of ischemic injury. CONCLUSIONS: The transient increases and selective tissue distribution of MCP-1 during early inflammation and muscle regeneration support the hypothesis that this cytokine participates in the early reparative events preceding the restoration of vascular perfusion following ischemic injury.

Assembly of TbetaRI:TbetaRII:TGFbeta ternary complex in vitro with receptor extracellular domains is cooperative and isoform-dependent.

Transforming growth factor-beta (TGFbeta) isoforms initiate signaling by assembling a heterotetrameric complex of paired type I (TbetaRI) and type II (TbetaRII) receptors on the cell surface. Because two of the ligand isoforms (TGFbetas 1, 3) must first bind TbetaRII to recruit TbetaRI into the complex, and a third (TGFbeta2) requires a co-receptor, assembly is known to be sequential, cooperative and isoform-dependent. However the source of the cooperativity leading to recruitment of TbetaRI and the universality of the assembly mechanism with respect to isoforms remain unclear. Here, we show that the extracellular domain of TbetaRI (TbetaRI-ED) binds in vitro with high affinity to complexes of the extracellular domain of TbetaRII (TbetaRII-ED) and TGFbetas 1 or 3, but not to either ligand or receptor alone. Thus, recruitment of TbetaRI requires combined interactions with TbetaRII-ED and ligand, but not membrane attachment of the receptors. Cell-based assays show that TbetaRI-ED, like TbetaRII-ED, acts as an antagonist of TGFbeta signaling, indicating that receptor-receptor interaction is sufficient to compete against endogenous, membrane-localized receptors. On the other hand, neither TbetaRII-ED, nor TbetaRII-ED and TbetaRI-ED combined, form a complex with TGFbeta2, showing that receptor-receptor interaction is insufficient to compensate for weak ligand-receptor interaction. However, TbetaRII-ED does bind with high affinity to TGFbeta2-TM, a TGFbeta2 variant substituted at three positions to mimic TGFbetas 1 and 3 at the TbetaRII binding interface. This proves both necessary and sufficient for recruitment of TbetaRI-ED, suggesting that the three different TGFbeta isoforms induce assembly of the heterotetrameric receptor complex in the same general manner.

Kinetic properties and metabolic contributions of yeast mitochondrial and cytosolic NADP+-specific isocitrate dehydrogenases.

To compare kinetic properties of homologous isozymes of NADP+-specific isocitrate dehydrogenase, histidine-tagged forms of yeast mitochondrial (IDP1) and cytosolic (IDP2) enzymes were expressed and purified. The isozymes were found to share similar apparent affinities for cofactors. However, with respect to isocitrate, IDP1 had an apparent Km value approximately 7-fold lower than that of IDP2, whereas, with respect to alpha-ketoglutarate, IDP2 had an apparent Km value approximately 10-fold lower than that of IDP1. Similar Km values for substrates and cofactors in decarboxylation and carboxylation reactions were obtained for IDP2, suggesting a capacity for bidirectional catalysis in vivo. Concentrations of isocitrate and alpha-ketoglutarate measured in extracts from the parental strain were found to be similar with growth on different carbon sources. For mutant strains lacking IDP1, IDP2, and/or the mitochondrial NAD+-specific isocitrate dehydrogenase (IDH), metabolite measurements indicated that major cellular flux is through the IDH-catalyzed reaction in glucose-grown cells and through the IDP2-catalyzed reaction in cells grown with a nonfermentable carbon source (glycerol and lactate). A substantial cellular pool of alpha-ketoglutarate is attributed to IDH function during glucose growth, and to both IDP1 and IDH function during growth on glycerol/lactate. Complementation experiments using a strain lacking IDH demonstrated that overexpression of IDP1 partially compensated for the glutamate auxotrophy associated with loss of IDH. Collectively, these results suggest an ancillary role for IDP1 in cellular glutamate synthesis and a role for IDP2 in equilibrating and maintaining cellular levels of isocitrate and alpha-ketoglutarate.

Influence of compartmental localization on the function of yeast NADP+-specific isocitrate dehydrogenases.

Three differentially compartmentalized isozymes of isocitrate dehydrogenase (mitochondrial IDP1, cytosolic IDP2, and peroxisomal IDP3) in the yeast Saccharomyces cerevisiae catalyze the NADP(+)-dependent oxidative decarboxylation of isocitrate to form alpha-ketoglutarate. These enzymes are highly homologous but exhibit some significant differences in physical and kinetic properties. To examine the impact of these differences on physiological function, we exchanged promoters and altered organellar targeting information to obtain expression of IDP2 and IDP3 in mitochondria and of IDP1 and IDP3 in the cytosol. Physiological function was assessed as complementation by mislocalized isozymes of defined growth defects of isocitrate dehydrogenase mutant strains. These studies revealed that the IDP isozymes are functionally interchangeable for glutamate synthesis, although mitochondrial localization has a positive impact on this function during fermentative growth. However, IDP2, whether located in mitochondria or in the cytosol, provided the highest level of defense against endogenous or exogenous oxidative stress.