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The homeodomain transcription factor Prep1 was previously shown to regulate insulin sensitivity. Our aim was to study the specific role of Prep1 for the regulation of energy metabolism in skeletal muscle. Muscle-specific ablation of Prep1 resulted in increased expression of respiratory chain subunits. This finding was consistent with an increase in mitochondrial enzyme activity without affecting mitochondrial volume fraction as assessed by electron microscopy. Metabolic phenotyping revealed no differences in daily energy expenditure or body composition. However, during treadmill exercise challenge, Prep1 ablation resulted in a higher maximal oxidative capacity and better endurance. Elevated PGC-1α expression was identified as a cause for increased mitochondrial capacity in Prep1 ablated mice. Prep1 stabilizes p160 Mybbp1a, a known inhibitor of PGC-1α activity. Thereby, p160 protein levels were significantly lower in the muscle of Prep1 ablated mice. By a chromatin immunoprecipitation-sequencing (ChIP-seq) approach, PREP1 binding sites in genes encoding mitochondrial components (e.g., Ndufs2) were identified that might be responsible for elevated proteins involved in oxidative phosphorylation (OXPHOS) in the muscle of Prep1 null mutants. These results suggest that Prep1 exhibits additional direct effects on regulation of mitochondrial proteins. We therefore conclude that Prep1 is a regulator of oxidative phosphorylation components via direct and indirect mechanisms.

Prep1 is a homeodomain transcription factor that acts by dimerizing with Pbx. Since Prep1 null embryos die at gastrulation, we studied Prep1(i/i) hypomorphic mice to study the physiological role of Prep1. A low percentage of homozygous Prep1(i/i) mice survived at birth, and their postnatal functions could be investigated. Reduced Prep1 expression caused an abnormal thymic T-cell development: increased CD4(-) CD8(-) double-negative thymocytes, decrease in alphabetaTCR(high) cells (cells with high levels of the alphabetaTau-cell receptor [alphabetaTCR]) and CD4(+) and CD8(+) single-positive (SP) thymocytes, and increase in gammadeltaTCR cells. Peripheral lymphoid organs of Prep1(i/i) mice contained fewer alphabetaTCR mature T cells and more gammadeltaTCR T cells than wild-type littermates. Moreover, Prep1(i/i) CD4(+) CD8(+) double-positive thymocytes underwent more apoptosis, and SP thymocytes proliferated less than control littermates. Mice that were lethally irradiated and then had Prep1(i/i) fetal liver cells transplanted showed the same defects as the Prep1(i/i) mice did. Among PBC family members, Pbx2 and very low levels of Pbx3 were observed in the thymi of wild-type mice. In Prep1(i/i) mice, the level of Pbx2 protein was profoundly decreased, while for Pbx3 no definitive conclusion could be reached. Therefore, the deficient postnatal T-lymphocytic potential of the Prep1 hematopoietic progenitors depends on the combined, not compensated, absence of Prep1 and at least Pbx2.

Extracellular matrix (ECM) formation and remodeling are critical processes for proper morphogenesis, organogenesis, and tissue repair. The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) inhibits ECM accumulation by stimulating the expression of matrix proteolytic enzymes and by downregulating the deposition of structural macromolecules such as type I collagen. Stimulation of ECM degradation has been linked to prolonged activation of jun gene expression by the cytokine. Here we demonstrate that TNF-alpha inhibits transcription of the gene coding for the alpha2 chain of type I collagen [alpha2(I) collagen] in cultured fibroblasts by stimulating the synthesis and binding of repressive CCAAT/enhancer proteins (C/EBPs) to a previously identified TNF-alpha-responsive element. This conclusion was based on the concomitant identification of C/EBPbeta and C/EBPdelta as TNF-alpha-induced factors by biochemical purification and expression library screening. It was further supported by the ability of the C/EBP-specific dominant-negative (DN) protein to block TNF-alpha inhibition of alpha2(I) collagen but not TNF-alpha stimulation of the MMP-13 protease. The DN protein also blocked TNF-alpha downregulation of the gene coding for the alpha1 chain of type I collagen. The study therefore implicates repressive C/EBPs in the TNF-alpha-induced signaling pathway that controls ECM formation and remodeling.