Conditional Deletion of Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Gene Reveals Its Essential Role in Chondrocyte Function and Endochondral Bone Formation

Abstract

The hypoxic growth plate cartilage requires hypoxia-inducible factor (HIF)-mediated pathways to maintain chondrocyte survival and differentiation. HIF proteins are tightly regulated by prolyl hydroxylase domain-containing protein 2 (Phd2)-mediated proteosomal degradation. We conditionally disrupted the Phd2 gene in chondrocytes by crossing Phd2 floxed mice with type 2 collagen-α1-Cre transgenic mice and found massive increases (>50%) in the trabecular bone mass of long bones and lumbar vertebra of the Phd2 conditional knockout (cKO) mice caused by significant increases in trabecular number and thickness and reductions in trabecular separation. Cortical thickness and tissue mineral density at the femoral middiaphysis of the cKO mice were also significantly increased. Dynamic histomorphometric analyses revealed increased longitudinal length and osteoid surface per bone surface in the primary spongiosa of the cKO mice, suggesting elevated conversion rate from hypertrophic chondrocytes to mineralized bone matrix as well as increased bone formation in the primary spongiosa. In the secondary spongiosa, bone formation measured by mineralizing surface per bone surface and mineral apposition rate were not changed, but resorption was slightly reduced. Increases in the mRNA levels of SRY (sex determining region Y)-box 9, osterix (Osx), type 2 collagen, aggrecan, alkaline phosphatase, bone sialoprotein, vascular endothelial growth factor, erythropoietin, and glycolytic enzymes in the growth plate of cKO mice were detected by quantitative RT-PCR. Immunohistochemistry revealed an increased HIF-1α protein level in the hypertrophic chondrocytes of cKO mice. Infection of chondrocytes isolated from Phd2 floxed mice with adenoviral Cre resulted in similar gene expression patterns as observed in the cKO growth plate chondrocytes. Our findings indicate that Phd2 suppresses endochondral bone formation, in part, via HIF-dependent mechanisms in mice.

Prolyl hydroxylase domain proteins regulate bone mass through their expression in osteoblasts

“…Suppression of PHD2 resulted in an up-regulation of SOX9 and enhanced extracellular matrix synthesis in human chondrocytes (Thoms and Murphy, 2010). Cheng et al reported conditional deletion of Phd2 in chondrocytes caused great increases in the trabecular bone mass of long bones and lumbar vertebra of mice (Cheng et al, 2016).…”

Conditional Deletion of the Phd2 Gene in Articular Chondrocytes Accelerates Differentiation and Reduces Articular Cartilage Thickness

“…Conditional deletion of HIF-1α in mesenchymal cells resulted in abnormal cartilage and joint development2021. On the other hand, we found that deletion of Phd2 in chondrocytes caused accelerated chondrocyte differentiation and increased endochondral bone formation in the metaphyses and epiphyses of long bones1314. These data clearly demonstrate that Phd2 negatively regulates chondrocyte differentiation and endochondral bone formation through the PHD/HIF regulatory pathway.…”

“…Prolyl Hydroxylase Domain-containing Proteins (PHDs) are negative regulators of the hypoxia-inducible factors (HIFs) which moderate chondrocyte differentiation131415. PHDs belong to the 2-oxoglutarate/iron-dependent dioxygenase superfamily.…”

The Roles and Mechanisms of Actions of Vitamin C in Bone: New Developments

“…A conditional knockout of PHD2 in collagen 2 positive (Col2 + ) chondrocytes leads to a dramatic increase in endochondral bone formation and trabecular bone volume at multiple skeletal sites. The increased bone formation is caused by increased chondrocyte and osteoblast functions as reflected by the marked increases in expression levels of osterix, ALP, and bone sialoprotein, as well as HIF1α and chondrogenic genes such as Col2 and Sox9 (95) . PHD2 inhibition in chondrocytes additionally increases the potential for cartilage repair by preventing degradation of HIF2α, which acts as an upstream enhancer of expression of the cartilage regulator Sox9 (96) .…”