Hypoxia suppresses runx2 independent of modeled microgravity

Abstract

Bone loss is a consequence of skeletal unloading as seen in bed rest and space flight. Unloading decreases oxygenation and osteoblast differentiation/function in bone. Previously we demonstrated that simulation of unloading in vitro, by culturing differentiating mouse osteoblasts in a horizontal rotating wall vessel (RWV), results in suppressed expression of runx2, a master transcriptional regulator of osteoblast differentiation. However, the RWV is able to reproduce in a controlled fashion at least two aspects of disuse that are directly linked, model microgravity and hypoxia. Hypoxia in the RWV is indicated by reduced medium oxygen tension and increased expression of GAPDH and VEGF. To uncouple the role of model microgravity from hypoxia in suppressed runx2 expression, we cultured osteoblasts under modeled microgravity (oxygenated, horizontal RWV rotation), hypoxia (vertical RWV rotation), or both conditions (horizontal RWV rotation). The expression, DNA binding activity and promoter activity of runx2, was suppressed under hypoxic but not normoxic modeled microgravity RWV conditions. Consistent with a role for hypoxia in suppression of runx2, direct exposure to hypoxia alone is sufficient to suppress runx2 expression in osteoblasts grown in standard tissue culture plates. Taken together, our findings indicate that hypoxia associated with skeletal unloading could be major suppressor of runx2 expression leading to suppressed osteoblast differentiation and bone formation.

Prolyl‐hydroxylase inhibition and HIF activation in osteoblasts promotes an adipocytic phenotype

“…Osteoblasts are one of the cell types in bone that are capable of responding to hypoxia. Previously, we and others have shown that runx2 and alkaline phosphatase (AP) expression (markers of osteoblast lineage selection and differentiation, respectively) are markedly suppressed in osteoblasts under hypoxic conditions Ontiveros et al, 2004;Salim et al, 2004]. These findings correspond with reports demonstrating that conditions of decreased bone oxygenation such as decreased arterial supply (chronic intramedullary pressure [Kiaer et al, 1992;Otter et al, 1999]) and inflammation (osteoarthritis [Kofoed, 1986;Kiaer et al, 1988]) are associated with bone loss.…”

“…While it is clear that hypoxia and HIFs are involved in osteoblast VEGF expression [Steinbrech et al, 2000;Akeno et al, 2001;Kim et al, 2002], less is known about how hypoxia and associated HIF induction regulate osteoblast phenotype and function. Previously, we reported that hypoxia suppresses runx2 mRNA levels in osteoblasts [Ontiveros et al, 2004]. Here we examine HIF and PHD expression in response to hypoxia, and the role of PHD and HIF activity in suppressing markers of osteoblast maturation and enhancing adipocyte phenotype.…”

“…AP and Runx2, genes associated with osteoblast differentiation, were analyzed by using HPRT as the internal control as previously described [Vengellur and LaPres, 2004;Botolin et al, 2005]. VEGF (a hypoxia-inducible gene) and C/EBPb, PPARg2 and aP2 (markers of adipocyte phenotype) were also measured with primers previously described [Ontiveros et al, 2004;Phan et al, 2004;Botolin et al, 2005]. Additional primer sets include HIF-1a (5 0 -TGG CTC CCT ATA TCC CAA TG-3 0 and 5 0 -GGT CTG CTG GAA CCC AGT AA-3 0 ), HIF-2a (5 0 -GAG CAA GCC TTC CAA GAC AC-3 0 and 5 0 -TTC GCA CTG ATG GTC TTG TC-3 0 ) and PHD1 (5 0 -GGA ACC CAC ATG AGG TGA AG-3 0 and 5 0 -AAC ACC TTT CTG TCC CGA TG-3 0 ), PHD2 (5 0 -GAA GCT GGG CAA CTA CAG GA-3 0 and 5 0 -CAT GTC ACG CAT CTT CCA TC-3 0 ), and PHD3 (5 0 -AAG TTA CAC GGA GGG GTC CT-3 0 and 5 0 -GGC TGG ACT TCA TGT GGA TT-3 0 ).…”

GATA-3 transduces survival signals in osteoblasts through upregulation of bcl-x L gene expression

“…The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were, respectively 5'-TTGGACAATGGACTGGTTG-3' and 3'-GTGACTGGTAGGTGAGATG-5' for Bcl-X L , 5'-CCTACC-GGGTTCGGATGTAA-3' and 3'-TGTCTTCCGTCCCTCACACAC-5' for GATA-3, 5'-GACAGAAGCTTGATGACTCTAAACC-3' and 5'-CTGTA-ATCTGACTCTGTCCTTGTG-3' for Runx2, and 5'-TATGGAGAA-GATTTGGCACC-3' and 3'-ATGAGACACACCTAACCACC-5' for bactin. (8,13,33) A real-time PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad).…”

Hypoxia mediates osteocyte ORP150 expression and cell death in vitro

“…It has been suggested that the loss of bone observed during disuse is the result of osteocyte hypoxia, resulting from unloading that reduces the interstitial fluid flow and, consequentially, reduces oxygen transport (18,19). It is widely regarded that disruption of the lacuno-canalicular network, which is necessary for nutrient and gaseous exchange for osteocytes, results in localized hypoxia in bone (20,21).…”