Prolyl Hydroxylase Domain-Containing Protein 3 Gene Expression in Chondrocytes Is Not Essential for Bone Development in Mice.

Weirong Xing,Sheila Pourteymoor,Subburaman Mohan,Gustavo A Gomez,Yian Chen

doi:10.3390/cells10092200

Weirong Xing, Sheila Pourteymoor + Show 3 more

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https://doi.org/10.3390/cells10092200

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Abstract

We previously showed that conditional disruption of the Phd2 gene in chondrocytes led to a massive increase in long bone trabecular bone mass. Loss of Phd2 gene expression or inhibition of PHD2 activity by a specific inhibitor resulted in a several-fold compensatory increase in Phd3 expression in chondrocytes. To determine if expression of PHD3 plays a role in endochondral bone formation, we conditionally disrupted the Phd3 gene in chondrocytes by crossing Phd3 floxed (Phd3flox/flox) mice with Col2α1-Cre mice. Loss of Phd3 expression in the chondrocytes of Cre+; Phd3flox/flox conditional knockout (cKO) mice was confirmed by real time PCR. At 16 weeks of age, neither body weight nor body length was significantly different in the Phd3 cKO mice compared to Cre−; Phd3flox/flox wild-type (WT) mice. Areal BMD measurements of total body as well as femur, tibia, and lumbar skeletal sites were not significantly different between the cKO and WT mice at 16 weeks of age. Micro-CT measurements revealed significant gender differences in the trabecular bone volume adjusted for tissue volume at the secondary spongiosa of the femur and the tibia for both genotypes, but no genotype difference was found for any of the trabecular bone measurements of either the femur or the tibia. Trabecular bone volume of distal femur epiphysis was not different between cKO and WT mice. Histology analyses revealed Phd3 cKO mice exhibited a comparable chondrocyte differentiation and proliferation, as evidenced by no changes in cartilage thickness and area in the cKO mice as compared to WT littermates. Consistent with the in vivo data, lentiviral shRNA-mediated knockdown of Phd3 expression in chondrocytes did not affect the expression of markers of chondrocyte differentiation (Col2, Col10, Acan, Sox9). Our study found that Phd2 but not Phd3 expressed in chondrocytes regulates endochondral bone formation, and the compensatory increase in Phd3 expression in the chondrocytes of Phd2 cKO mice is not the cause for increased trabecular bone mass in Phd2 cKO mice.

Highlights

IntroductionAll three prolyl hydroxylase domain (PHD) enzymes including PHD1, PHD2 and PHD3 share a highly conserved hydroxylase domain in the catalytic C-terminal regions, whereas the N-terminal regions are more divergent and with no known functions [1]
In our studies on the mechanism by which PHD2 regulates chondrocyte differentiation and increased bone formation, we found that loss of PHD2 function in chondrocytes resulted in marked upregulation of Phd3 expression, both in vitro and in vivo
To test whether Phd3 is disrupted in the bones of conditional knockout (cKO) mice, total RNA was extracted from the distal femur epiphysis and growth bones of cKO mice, total RNA was extracted from the distal femur epiphysis and growth plate region of 16-week old cKO and WT mice and used for real-time PCR with specific plate region of 16-week old cKO and WT mice and used for real-time PCR with specific primers

Summary

Introduction

All three prolyl hydroxylase domain (PHD) enzymes including PHD1, PHD2 and PHD3 share a highly conserved hydroxylase domain in the catalytic C-terminal regions, whereas the N-terminal regions are more divergent and with no known functions [1]. Both PHD1 and PHD2 contain more than 400 amino acid residues, while PHD3 has less than 250 with a short N-terminal sequence. The hydroxylation of specific proline residues (Pro-402 and Pro-564) in the oxygen-dependent degradation domains (ODDs) of the HIF1α by PHDs leads to the targeting of HIF1α for ubiquitination through an E3 ligase complex initiated by the binding of the

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