Mice deficient in the SDF1-chemokine-receptor CXCR4, exhibit severe defects of secondary limb myogenesis. To further elucidate the role of SDF1 in muscle development, we have now analyzed putative effects of this chemokine on proliferation, migration and myogenic differentiation of mouse C2C12 myogenic progenitor/myoblast cells. In addition, we have characterized the signaling pathways employed by SDF1-CXCR4 to control myogenesis. We found that SDF1 stimulates proliferation and induces migration of C2C12 cells with a potency similar to that of FGF2 and HGF, which both represent prototypical extracellular regulators of myogenesis. In addition, SDF1 inhibits myogenic differentiation in both C2C12 cells and primary myoblasts, as assessed by MyoD, myosin heavy chain and/or myogenin expression. Regarding signaling pathways, C2C12 cells responded to SDF1 with activation (phosphorylation) of Erk and PKCζ, whereas even after prolonged SDF1 treatment for up to 120 minutes, levels of activated Akt, p38 and PKCα or PKCβ remained unaffected. Preventing activation of the classic MAP kinase cascade with the Erk inhibitor UO126 abolished SDF1-induced proliferation and migration of C2C12 cells but not the inhibitory action of SDF1 on myogenic differentiation. Moreover, the effects of SDF1 on proliferation, migration and differentiation of C2C12 cells were all abrogated in the presence of myristoylated PKCζ peptide pseudosubstrate and/or upon cellular depletion of PKCζ by RNA interference. In conclusion, our findings unravel a previously unknown role of CXCR4-PKCζ signaling in myogenesis. The potent inhibitory effects of SDF1 on myogenic differentiation point to a major function of CXCR4-PKCζ signaling in the control of secondary muscle growth.

SummaryThe alternative SDF-1 (stromal cell derived factor-1) receptor, CXCR7, has been suggested to act as either a scavenger of extracellular SDF-1 or a modulator of the primary SDF-1 receptor, CXCR4. CXCR7, however, also directly affects the function of various tumor-cell types. Here, we demonstrate that CXCR7 is an active component of SDF-1 signalling in astrocytes and Schwann cells. Cultured cortical astrocytes and peripheral nerve Schwann cells exhibit comparable total and cell-surface levels of expression of both SDF-1 receptors. Stimulation of astrocytes with SDF-1 resulted in the temporary activation of Erk1/2, Akt and PKC/, but not p38 and PKC/. Schwann cells showed SDF-1-induced activation of Erk1/2, Akt and p38, but not PKC/ and PKC/. The respective signalling pattern remained fully inducible in astrocytes from CXCR4-deficient mice, but was abrogated following depletion of astrocytic CXCR7 by RNAi. In Schwann cells, RNAi-mediated depletion of either CXCR4 or CXCR7 silenced SDF-1 signalling. The findings of the astrocytic receptor-depletion experiments were reproduced by CXCR7 antagonist CCX754, but not by CXCR4 antagonist AMD3100, both of which abolished astrocytic SDF-1 signalling. Further underlining the functional importance of CXCR7 signalling in glial cells, we show that the mitogenic effects of SDF-1 on both glial cell types are impaired upon depleting CXCR7.

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