Endochondral ossification, an important bone formation process in vertebrates, highly depends on proper functioning of growth plate chondrocytes 1 . Their proliferation determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment 1,2 . In addition, the centre of the expanding growth plate becomes hypoxic and local activation of the hypoxiainducible transcription factor HIF-1α is necessary for chondrocyte survival by still unknown cellintrinsic mechanisms [3][4][5][6] . Whether HIF-1α signalling has to be contained in the other regions of the growth plate and whether chondrocyte metabolism controls cell function remains undefined. We here show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response (UPR) and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate (αKG) levels, which in turn increases collagen proline and lysine hydroxylation. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to proteasemediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other extracellular matrix-related diseases such as cancer and fibrosis.To investigate whether HIF signalling needs to be controlled in growth plate chondrocytes, we conditionally inactivated HIF prolyl hydroxylase 2 (PHD2; Phd2 chonmice), its main negative regulator 7 , resulting in HIF-1α accumulation (Extended Data Fig. 1a-d).This approach caused skeletal dysplasia, characterized by impaired longitudinal bone growth and increased trabecular bone mass (Fig. 1a,b, Extended Data Fig. 1e,f). The growth plate was shorter, but normally organized and, interestingly, the high bone mass was not due to altered bone resorption or formation (Extended Data Fig. 1g-l). Instead, we observed more cartilage remnants in the bony trabeculae, evidenced by more type II collagen (COL2)positive and proteoglycan-rich matrix (Fig. 1c, Extended Data Fig. 1m). The decreased serum CTx-II levels, measuring COL2 degradation, indicated that the cartilage matrix was incompletely resorbed, and the unaltered chondrocyte-to-matrix ratio pointed to a qualitative, rather than quantitative, change in matrix properties (Extended Data Fig. 1j,n). Thus, inactive oxygen sensing in chondrocytes increases trabecular bone mass, caused by abundant cartilage remnants, likely resulting from modifications in the cartilage matrix itself.HIF-1α stabilization in PHD2-deficient chondrocytes resulted, as expected 7,8 , in metabolic reprogramming. Mitochondrial content was reduced, likely because of decreased biogenesis without changing autophagy (Extended D...

Edited by Xiao-Fan WangTandem mass spectrometry was applied to tissues from targeted mutant mouse models to explore the collagen substrate specificities of individual members of the prolyl 3-hydroxylase (P3H) gene family. Previous studies revealed that P3h1 preferentially 3-hydroxylates proline at a single site in collagen type I chains, whereas P3h2 is responsible for 3-hydroxylating multiple proline sites in collagen types I, II, IV, and V. In screening for collagen substrate sites for the remaining members of the vertebrate P3H family, P3h3 and Sc65 knock-out mice revealed a common lysine under-hydroxylation effect at helical domain cross-linking sites in skin, bone, tendon, aorta, and cornea. No effect on prolyl 3-hydroxylation was evident on screening the spectrum of known 3-hydroxyproline sites from all major tissue collagen types. However, collagen type I extracted from both The collagen family of proteins has evolved into many different genes and gene translational products each with variably regulated post-translational modifications (1, 2). Even within a single genetic type of collagen, the post-translational quality of the protein is known to be regulated with a high degree of tissue specificity (3, 4). The functional significance of this post-translational variability is still not fully understood, although for fibril-forming collagens the number, placement, and chemistry of covalent intermolecular cross-links seem to be critically important regulators of tissue material properties and function (5-7).In the last decade, new insights on the significance of a relatively rare collagen modification, prolyl 3-hydroxylation, came from the discovery that recessive forms of osteogenesis imperfecta (OI) 2 are caused by biallelic mutations in prolyl 3-hydroxylase 1 (P3H1; Lepre1), CRTAP (Leprel3), or CypB (PPIB) (8). These proteins, which form a P3H1 enzyme complex, act on nascent collagen chains in the ER (9). Mutations in at least 6 further genes that encode either enzymes (e.g. PLOD2 encoding lysyl hydroxylase-2 (LH2) (10)) or chaperones (e.g. FKBP10 encoding FKBP65 (11, 12)), needed for collagen modification, folding, transport, and normal mineralization, have been shown to cause OI variants. We have gained insights on the disease-causing mechanisms by analyzing tissue collagens from OI patients and mouse models of OI. For example, P3H1, CRTAP, and PPIB mutations all cause telopeptide lysine overhydroxylation (5), whereas PLOD2 or FKBP10 mutations cause telopeptide lysine under-hydroxylation (10 -12). A common effect from all these mutations is an altered collagen cross-linking chemistry. These findings suggest an interplay in the ER between the prolyl 3-hydroxylation complex and the lysyl hydroxylation machinery.Human mutations in P3H2 (Leprel1) have been shown to cause the eye disorder high myopia (13-15). Two different P3H2 knock-out mice have so far been generated by separate laboratories, the first was embryonic lethal (16), whereas the second had no obvious phenotype (17). Mass spectral analysis of...

Background:Collagen cross-linking mechanisms must be regulated to obtain tissue-specific collagen fiber properties. Results: Deficiency in collagen-associated protein fibromodulin leads to excessively cross-linked specific domain of collagen. Conclusion: Fibromodulin modulates site-specific cross-linking of collagen. Significance: This is the first report showing that a collagen-associated protein can modulate cross-linking of specific collagen domains.

Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis.