Reduced Bone Mass in Collagen Prolyl 4‐Hydroxylase P4ha1^+/−; P4ha2^−/− Compound Mutant Mice

Abstract

Proper deposition of the extracellular matrix and its major components, the collagens, is essential for endochondral ossification and bone mass accrual. Collagen prolyl 4‐hydroxylases (C‐P4Hs) hydroxylate proline residues in the ‐X‐Pro‐Gly‐ repeats of all known collagen types. Their product, 4‐hydroxyproline, is essential for correct folding and thermal stability of the triple‐helical collagen molecules in physiological body temperatures. We have previously shown that inactivation of the mouse P4ha1 gene, which codes for the catalytic α subunit of the major C‐P4H isoform, is embryonic lethal, whereas inactivation of the P4ha2 gene produced only a minor phenotype. Instead, mice with a haploinsufficiency of the P4ha1 gene combined with a homozygous deletion of the P4ha2 gene present with a moderate chondrodysplasia due to transient cell death of the growth plate chondrocytes. Here, to further characterize the bone phenotype of the P4ha1+/−; P4ha2−/− mice, we have carried out gene expression analyses at whole‐tissue and single‐cell levels, biochemical analyses, microcomputed tomography, histomorphometric analyses, and second harmonic generation microscopy to show that C‐P4H α subunit expression peaks early and that the C‐P4H deficiency leads to reduced collagen amount, a reduced rate of bone formation, and a loss of trabecular and cortical bone volume in the long bones. The total osteoblast number in the proximal P4ha1+/−; P4ha2−/− tibia and the C‐P4H activity in primary P4ha1+/−; P4ha2−/− osteoblasts were reduced, whereas the population of osteoprogenitor colony‐forming unit fibroblasts was increased in the P4ha1+/−; P4ha2−/− marrow. Thus, the P4ha1+/−; P4ha2−/− mouse model recapitulates key aspects of a recently recognized congenital connective tissue disorder with short stature and bone dysplasia caused by biallelic variants of the human P4HA1 gene. Altogether, the data demonstrate the allele dose‐dependent importance of the C‐P4Hs to the developing organism and a threshold effect of C‐P4H activity in the proper production of bone matrix. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Collagen prolyl 4-hydroxylases have sequence specificity towards different X-Pro-Gly triplets

“…to have some differences in expression levels and tissue distribution (Annunen et al, 1997(Annunen et al, , 1998Tolonen et al, 2022), although also strikingly similar patterns have been described (Helaakoski et al, 1995). Our expression data showed that P4ha1 is the most abundant isoform in mouse skin, kidney and in MEFs (Figs 1A, 2A, 3A).…”

“…However, these genetic deletions have very different consequences for life. The P4ha1 null mice exhibit a massive type IV collagen defect and early embryonic lethality (Holster et al, 2007) in contrast to P4ha2 knockout mice, which develop to term and the adult mutant mice are only mildly affected (Aro et al, 2015;Tolonen et al, 2022).…”

“…P4ha2 knockout mice have no overt phenotype and only minor abnormalities in their tissues and extracellular matrix have been described (Aro et al, 2015;Tolonen et al, 2022). However, combining complete lack of C-P4H-II with reduced C-P4H-I amount (P4ha1 +/-;P4ha2 -/mice) leads to chondrodysplasia and extracellular matrix defects in many tissues (Aro et al, 2015;Tolonen et al, 2022).…”

Tissue-specific collagen hydroxylation at GEP/GDP triplets mediated by P4HA2

“…The general inhibition of all three P4HAs results in a very distinct hydroxylation profile (figure 4), suggesting that P4HA2 is better able than P4HA1 and P4HA3 of hydroxylating GEP/GDPs. P4HA3 is poorly characterized yet, but P4HA1 and P4HA2 were so far believed to possess no intrinsic GXP substrate specificity [27,29]. Indeed, most GEP/GDPs exhibit significant, but not binary regulation of hydroxylation, in P4ha2-invalidated versus control cells (figure 3.…”

“…Their respective functional specificities remain elusive [27][28][29]. In mice, the P4ha1 knock-out is embryonically lethal [30], while that of P4ha2 has no major phenotype [30] and that of P4ha3 has not been reported yet.…”