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

SummaryObjectiveTo investigate the chondrogenic potential of growth factor-stimulated periosteal cells with respect to the activity of Hypoxia-inducible Factor 1α (HIF-1α).MethodsScaffold-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-1α 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-1α expression was assessed in vitro by employing specific inhibitors.ResultsUnstimulated 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-1α staining in superficial areas, but moderate HIF-1α 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-1α staining throughout all zones of the repair tissue and formed a hyaline-like matrix. In vitro, BMP-2 and IGF-1 supplementation increased HIF-1α protein levels in periosteal cells, which was based on posttranscriptional mechanisms rather than de novo mRNA synthesis, involving predominantly the MEK/ERK pathway.ConclusionThis 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-1α activity

ARHGEF9 disease: Phenotype clarification and genotype-phenotype correlation.

ObjectiveWe aimed to generate a review and description of the phenotypic and genotypic spectra of ARHGEF9 mutations.MethodsPatients with mutations or chromosomal disruptions affecting ARHGEF9 were identified through our clinics and review of the literature. Detailed medical history and examination findings were obtained via a standardized questionnaire, or if this was not possible by reviewing the published phenotypic features.ResultsA total of 18 patients (including 5 females) were identified. Six had de novo, 5 had maternally inherited mutations, and 7 had chromosomal disruptions. All females had strongly skewed X-inactivation in favor of the abnormal X-chromosome. Symptoms presented in early childhood with delayed motor development alone or in combination with seizures. Intellectual disability was severe in most and moderate in patients with milder mutations. Males with severe intellectual disability had severe, often intractable, epilepsy and exhibited a particular facial dysmorphism. Patients with mutations in exon 9 affecting the protein's PH domain did not develop epilepsy.ConclusionsARHGEF9 encodes a crucial neuronal synaptic protein; loss of function of which results in severe intellectual disability, epilepsy, and a particular facial dysmorphism. Loss of only the protein's PH domain function is associated with the absence of epilepsy

Renal tubular HIF-2α expression requires VHL inactivation and causes fibrosis and cysts.

The Hypoxia-inducible transcription Factor (HIF) represents an important adaptive mechanism under hypoxia, whereas sustained activation may also have deleterious effects. HIF activity is determined by the oxygen regulated α-subunits HIF-1α or HIF-2α. Both are regulated by oxygen dependent degradation, which is controlled by the tumor suppressor "von Hippel-Lindau" (VHL), the gatekeeper of renal tubular growth control. HIF appears to play a particular role for the kidney, where renal EPO production, organ preservation from ischemia-reperfusion injury and renal tumorigenesis are prominent examples. Whereas HIF-1α is inducible in physiological renal mouse, rat and human tubular epithelia, HIF-2α is never detected in these cells, in any species. In contrast, distinct early lesions of biallelic VHL inactivation in kidneys of the hereditary VHL syndrome show strong HIF-2α expression. Furthermore, knockout of VHL in the mouse tubular apparatus enables HIF-2α expression. Continuous transgenic expression of HIF-2α by the Ksp-Cadherin promotor leads to renal fibrosis and insufficiency, next to multiple renal cysts. In conclusion, VHL appears to specifically repress HIF-2α in renal epithelia. Unphysiological expression of HIF-2α in tubular epithelia has deleterious effects. Our data are compatible with dedifferentiation of renal epithelial cells by sustained HIF-2α expression. However, HIF-2α overexpression alone is insufficient to induce tumors. Thus, our data bear implications for renal tumorigenesis, epithelial differentiation and renal repair mechanisms

Gene array of VHL mutation and hypoxia shows novel hypoxia-induced genes and that cyclin D1 is a VHL target gene

Gene expression analysis was performed on a human renal cancer cell line (786-0) with mutated VHL gene and a transfectant with wild-type VHL to analyse genes regulated by VHL and to compare with the gene programme regulated by hypoxia. There was a highly significant concordance of the global gene response to hypoxia and genes suppressed by VHL. Cyclin D1 was the most highly inducible transcript and 14-3-3 epsilon was downregulated. There were some genes regulated by VHL but not hypoxia in the renal cell line, suggesting a VHL role independent of hypoxia. However in nonrenal cell lines they were hypoxia regulated. These included several new pathways regulated by hypoxia, including RNase 6PL, collagen type 1 alpha 1, integrin alpha 5, ferritin light polypeptide, JM4 protein, transgelin and L1 cell adhesion molecule. These were not found in a recent SAGE analysis of the same cell line. Hypoxia induced downregulation of Cyclin D1 in nonrenal cells via an HIF independent pathway. The selective regulation of Cyclin D1 by hypoxia in renal cells may therefore contribute to the tissue selectivity of VHL mutation

RNF4 and VHL regulate the proteasomal degradation of SUMO-conjugated Hypoxia-Inducible Factor-2α

Hypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1α and HIF-2α are continuously synthesized in cells and degraded via the ubiquitin–proteasome pathway. During hypoxia, these proteins are stabilized and translocate to the nucleus to activate transcription of target genes that enable cell survival at reduced oxygen levels. HIF proteins are tightly regulated via post-translational modifications including phosphorylation, acetylation, prolyl-hydroxylation and ubiquitination. Here we show for the first time that exogenous and endogenous HIF-2α are also regulated via the ubiquitin-like modifier small ubiquitin-like modifiers (SUMO). Using mutational analysis, we found that K394, which is situated in the sumoylation consensus site LKEE, is the major SUMO acceptor site in HIF-2α. Functionally, sumoylation reduced the transcriptional activity of HIF-2α. Similar to HIF-1α, HIF-2α is regulated by the SUMO protease SENP1. The proteasome inhibitor MG132 strongly stabilized SUMO-2-conjugated HIF-2α during hypoxia but did not affect the total level of HIF-2α. The ubiquitin E3 ligases von Hippel–Lindau and RNF4 control the levels of sumoylated HIF-2α, indicating that sumoylated HIF-2α is degraded via SUMO-targeted ubiquitin ligases

In vivo Identification and Specificity assessment of mRNA markers of hypoxia in human and mouse tumors

<p>Abstract</p> <p>Background</p> <p>Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1α target genes in vivo has been questioned due to the confounding influence of other microenvironmental abnormalities known to affect gene expression (e.g., low pH). Here we describe a new technique that by exploiting intratumoral oxygenation heterogeneity allows us to identify and objectively rank the most robust mRNA hypoxia biomarkers.</p> <p>Methods</p> <p>Mice carrying human (FaDu_dd) or murine (SCCVII) tumors were injected with the PET hypoxia tracer FAZA. Four hours post-injection tumors were removed, frozen, and crushed into milligram-sized fragments, which were transferred individually to pre-weighed tubes containing RNAlater and then weighed. For each fragment radioactivity per tissue mass and expression patterns of selected mRNA biomarkers were analyzed and compared.</p> <p>Results</p> <p>In both tumour models, fragmentation into pieces weighing 10 to 60 mg resulted in tissue fragments with highly variable relative content of hypoxic cells as evidenced by an up to 13-fold variation in FAZA radioactivity per mass of tissue. Linear regression analysis comparing FAZA retention with patterns of gene expression in individual tissue fragments revealed that CA9, GLUT1 and LOX mRNA levels were equally and strongly correlated to hypoxic extent in FaDu_dd. The same link between hypoxia and gene expression profile was observed for CA9 and GLUT1, but not LOX, in SCCVII tumors. Apparent in vivo hypoxia-specificity for other putative molecular markers of tissue hypoxia was considerably weaker.</p> <p>Conclusions</p> <p>The portrayed technique allows multiple pairwise measurements of mRNA transcript levels and extent of hypoxia in individual tumors at a smallest possible volumetric scale which (by limiting averaging effects inherent to whole-tumor analysis) strengthen the conclusiveness on true hypoxia-specificity of candidate genes while limiting the required number of tumors. Among tested genes, our study identified CA9, GLUT1 and possibly LOX as highly specific biomarkers of tumor hypoxia in vivo.</p