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Previously \nwe identified endogenous hydrogen peroxide-induced poly(ADP-\nribose) polymerase-1 (PARP1) activation as a mediator of \nosteodifferentiation and associated cell death. Here we set out \nto investigate whether or not activation of PARP1 is dependent \non DNA breaks and how PARP1 mediates cell death during \nosteodifferentiation of mesenchymal stem cells and SAOS-2 cells. \nHere we show that the MAP kinases p38, JNK, and ERK1/2 become \nactivated during the differentiation process. However, only p38 \nactivation depended both on hydrogen peroxide production and on \nPARP1 activation as the hydrogen peroxide decomposing enzyme \ncatalase, the PARP inhibitor PJ34, and the silencing of PARP1 \nsuppressed p38 activation. Inhibition of p38 suppressed cell \ndeath and inhibited osteogenic differentiation (calcium \ndeposition, alkaline phosphatase activity, and marker gene \nexpression) providing further support for the close coupling of \nosteodifferentiation and cell death. Metabolic collapse appears \nto be central in the hydrogen peroxide-PARP1-p38 pathway as \nsilencing PARP1 or inhibition of p38 prevented differentiation-\nassociated loss of cellular NAD, inhibition of mitochondrial \nrespiration, and glycolytic activity. We also provide evidence \nthat endogenous hydrogen peroxide produced by the \ndifferentiating cells is sufficient to cause detectable DNA \nbreakage. Moreover, p38 translocates from the cytoplasm to the \nnucleus where it interacts and colocalizes with PARP1 as \ndetected by immunoprecipitation and immunofluorescence, \nrespectively. 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