Chondrogenic differentiation of growth factor-stimulated precursor cells in cartilage repair tissue is associated with increased HIF-1alpha activity.

OBJECTIVE: To investigate the chondrogenic potential of growth factor-stimulated periosteal cells with respect to the activity of Hypoxia-inducible Factor 1alpha (HIF-1alpha). METHODS: Scaffold-bound autologous periosteal cells, which had been activated by Insulin-like Growth Factor 1 (IGF-1) or Bone Morphogenetic Protein 2 (BMP-2) gene transfer using both adeno-associated virus (AAV) and adenoviral (Ad) vectors, were applied to chondral lesions in the knee joints of miniature pigs. Six weeks after transplantation, the repair tissues were investigated for collagen type I and type II content as well as for HIF-1alpha expression. The functional role of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling on BMP-2/IGF-1-induced HIF-1alpha expression was assessed in vitro by employing specific inhibitors. RESULTS: Unstimulated periosteal cells formed a fibrous extracellular matrix in the superficial zone and a fibrocartilaginous matrix in deep zones of the repair tissue. This zonal difference was reflected by the absence of HIF-1alpha staining in superficial areas, but moderate HIF-1alpha expression in deep zones. In contrast, Ad/AAVBMP-2-stimulated periosteal cells, and to a lesser degree Ad/AAVIGF-1-infected cells, adopted a chondrocyte-like phenotype with strong intracellular HIF-1alpha staining throughout all zones of the repair tissue and formed a hyaline-like matrix. In vitro, BMP-2 and IGF-1 supplementation increased HIF-1alpha protein levels in periosteal cells, which was based on posttranscriptional mechanisms rather than de novo mRNA synthesis, involving predominantly the MEK/ERK pathway. CONCLUSION: This pilot experimental study on a relatively small number of animals indicated that chondrogenesis by precursor cells is facilitated in deeper hypoxic zones of cartilage repair tissue and is stimulated by growth factors which enhance HIF-1alpha activity.

Identification of a putative p53 binding sequence within the human mitochondrial genome.

A small fraction of the total cellular amount of nuclear transcription factor p53 seems to be located at and within mitochondria. Transcription factors of the steroid receptor superfamily that, like p53, lack a classical mitochondrial leader sequence are nonetheless imported into mitochondria where they regulate mtDNA transcription through binding to specific recognition sequences. Here, we examined seven candidate sequences from the human mitochondrial genome with similarity to the consensus p53 binding motif. Two imperfect half-sites at coordinate 1553 with homology to the nuclear IGF-BP3 box A binding sequence are demonstrated to confer responsivity to p53 and the p53 relatives p73alpha and beta in the context of the cell nucleus. Mitochondrial p53 may thus bind directly to mtDNA and, perhaps, be involved in the regulation of mitochondrial transcription/replication.

Cell type-specific regulation of calmodulin 2 expression by mutant p53.

To identify genes that are stimulated by oncogenic forms of mutant p53, we studied, by microarray analysis and PCR-select subtractive hybridization, gene expression changes in human wild-type (wt) p53-negative immortal 041 fibroblasts infected to stably express p53 mutant 175H. In contrast to the wt p53 transactivator, 175H induced only few and weak, gene expression changes. We report here the stimulation of calmodulin 2 (CaM 2), but not CaM 1 or 3, gene expression specifically in 041 cells. The stimulation of the CaM 2 promoter required the 5' untranslated sequences as well as the integrity of the transactivation domain of 175H. However, direct binding of 175H to the 5'UT in vitro could not be demonstrated.