br Materials and methods br Results br Discussion To
Materials and methods
Discussion To fulfill its role in tissue homeostasis, repair and immunoregulation, MSCs must be able to (i) renew themselves by cell division and proliferation, (ii) migrate to its target end-site in response to chemotaxis signals and (iii) undergo appropriate and effective cellular differentiation . The SDF-1/CXCR4 axis is a key regulator in modulating MSC migration. Activation of CXCR4 upon SDF-1 binding is mediated by the uncoupling of the intracellular heterotrimeric G-protein. Specific to its role in regulating chemotaxis, the dimeric Gβγ subunit activates several focal adhesion components including FAK and Paxillin by phosphorylation. Alternatively, chemotaxis can also be mediated via mitogen-activated protein kinases (MAPK) through the Gα-monomer, which can signal through ERK-1/2. Activation of either of these pathways results in cytoskeleton reorganization and cell migration [35,37,38]. We confirmed that the binding of SDF-1 to CXCR4 in MSCs resulted in activation of the both FAK and ERK pathways, which in turn enhanced cytoskeleton reorganization, with resultant MSC chemotaxis. Interestingly, inhibition of FAK by Y11 or PF573228 completely abolished ERK activation, but not vice versa. This suggests the presence of cross talk between these two signaling pathways, and FAK may be positioned upstream of ERK. Indeed, there have been similar reports of FAK acting as an adapter protein whose kinase activity is instrumental for the activation of ERK-1/2 [, , ]. Inhibition of either kinase resulted in failure of Palosuran stress fiber assembly and cell migration, thus supporting the importance of both kinases in SDF-1 induced chemotaxis in MSCs. HIV gp120 is known to downregulate the expression and function of CXCR4 in monocytes, B cells and T cells [, , ]. Surprisingly, we found that gp120 significantly upregulated the expression of CXCR4 in MSCs. We also demonstrated the functional import of gp120 in driving SDF-1/CXCR4 mediated MSC chemotaxis. Firstly, MSCs primed with gp120 showed increased migration toward SDF-1 compared to unprimed controls; this was blocked by the CXCR4 antagonist AMD3100. Secondly, in gp120-primed MSCs, there was over-activation of FAK, ERK and enhanced actin stress fiber assembly with concurrent CXCR4 upregulation. Finally, in an siRNA-mediated CXCR4 knockdown cell model, both SDF-1 induced and gp120-enhanced activation of FAK and the assembly of actin stress fibers were abrogated, with resultant inhibition of cell migration. The decision for pluripotent MSCs to commit to a specific cellular lineage is thought to be under the control of a series of complex and coordinated cellular signaling pathways, with the end result of effective differentiation being appropriate to the tissue homeostasis and regeneration requirements. Several groups have already reported disruption in MSC differentiation due to HIV infection and/or its proteins. HIV proteins and serum from HIV-infected individuals can preferentially drive MSC differentiation toward a pro-adipogenic phenotype, thus potentially contributing to dyslipidemia. In parallel, there was downregulation of the osteogenic process, which putatively contributes to reduced bone formation and osteoporosis [11,12]. A similar pro-adipogenic differentiation process was described in vascular wall derived MSCs . This may in turn contribute to increased incidence of atherosclerosis and cardiovascular disease in HIV-positive individuals. We postulate that the increased gp120-associated MSC migration described in this study, further compounds the problem by mobilizing the MSC population destined for ineffective and inappropriate differentiation commitment. As HIV-entry co-receptors, CXCR4 and CCR5 are physically associated with CD4 receptors; the HIV fusion reaction is initiated by sequential receptor binding of the HIV surface glycoprotein gp120 to CD4, then to either CXCR4 (T cell line tropic HIV strains, T-tropic) or CCR5 (macrophage-tropic, M-tropic HIV strains) on susceptible human cells [42,43]. R5 variants are generally seen in early stages of HIV infection and are believed to be more efficient in HIV transmission and establishment of infection. X4 variants tend to emerge in later stages of HIV infection, and are associated with more severe pathogenicity, immunosuppression and rapid progression to AIDS [, , ]. The aetiology of this switch in dominant viral co-receptor tropism in the natural history of HIV infection remains unclear. In the present study, gp120 from both T- and M-tropic HIV strains induced similar upregulation of CXCR4 in MSCs. Given that MSCs may be permissive to HIV infection [11,17], we speculate that initial cellular priming by gp120 results in increased expression of CXCR4 and this may lead to co-receptor switching and cell tropism changes in chronic HIV infection.