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Abstract NAD is an essential co-factor for cellular energy metabolism and multiple other processes. Systemic NAD+ deficiency has been implicated in skeletal deformities during development in both humans and mice. NAD levels are maintained by multiple synthetic pathways but which ones are important in bone forming cells is unknown. Here, we generate mice with deletion of Nicotinamide Phosphoribosyltransferase (Nampt), a critical enzyme in the NAD salvage pathway, in all mesenchymal lineage cells of the limbs. At birth, Nampt ΔPrx1 exhibit dramatic limb shortening due to death of growth plate chondrocytes. Administration of the NAD precursor nicotinamide riboside during pregnancy prevents the majority of in utero defects. Depletion of NAD post-birth also promotes chondrocyte death, preventing further endochondral ossification and joint development. In contrast, osteoblast formation still occurs in knockout mice, in line with distinctly different microenvironments and reliance on redox reactions between chondrocytes and osteoblasts. These findings define a critical role for cell-autonomous NAD homeostasis during endochondral bone formation.

Specific small molecule RXFP2 agonists with favorable pharmacokinetic properties induce gubernacular invagination in mouse embryos, increase mineralization activity in human osteoblasts in vitro, and improve bone trabecular parameters in adult mice.

Smith McCort (SMC) dysplasia is a rare, autosomal recessive, osteochondrodysplasia that can be caused by pathogenic variants in either RAB33B or DYM genes. These genes codes for proteins that are located at the Golgi apparatus and have a role in intracellular vesicle trafficking. We generated mice that carry a Rab33b disease-causing variant, c.136A>C (p.Lys46Gln), which is identical to that of members from a consanguineous family diagnosed with SMC. In male mice at 4 months of age, the Rab33b variant caused a mild increase in trabecular bone thickness in the spine and femur and in femoral mid-shaft cortical thickness with a concomitant reduction of the femoral medullary area, suggesting a bone resorption defect. In spite of the increase in trabecular and cortical thickness, bone histomorphometry showed a 4-fold increase in osteoclast parameters in homozygous Rab33b mice suggesting a putative impairment in osteoclast function, while dynamic parameters of bone formation were similar in mutant versus control mice. Femur biomechanical tests showed an increased in yield load and a progressive elevation, from WT to heterozygote to homozygous mutants, of bone intrinsic properties. These findings suggest an overall impact on bone material properties which may be caused by disturbed protein glycosylation in cells contributing to skeletal formation, supported by the altered and variable pattern of lectin staining in murine and human tissue cultured cells and in liver and bone murine tissues. The mouse model only reproduced some of the features of the human disease and was sex-specific, manifesting in male but not female mice. Our data reveal a potential novel role of RAB33B in osteoclast function and protein glycosylation and their dysregulation in SMC and lay the foundation for future studies.

Abstract The Ehlers‐Danlos syndromes (EDS) are inherited connective tissue diseases with primary manifestations that affect the skin and the musculoskeletal system. However, the effects of EDS on the respiratory system are not well understood and are described in the literature as sporadic case reports. We performed histological, histomorphometric, and the first in‐depth characterization of respiratory system function in a mouse model of classical EDS (cEDS) with haploinsufficiency of type V collagen (Col5a1+/−). In young adult male and female mice, lung histology showed reduced alveolar density, reminiscent of emphysematous‐like changes. Respiratory mechanics showed a consistent increase in respiratory system compliance accompanied by increased lung volumes in Col5a1+/− compared to control mice. Flow–volume curves, generated to mimic human spirometry measurements, demonstrated larger volumes throughout the expiratory limb of the flow volume curves in Col5a1+/− compared to controls. Some parameters showed a sexual dimorphism with significant changes in male but not female mice. Our study identified a clear respiratory phenotype in the Col5a1+/− mouse model of EDS and indicated that intrinsic respiratory and lung changes may exist in cEDS patients. Their potential impact on the respiratory function during lung infections, other respiratory disease processes, or insults may be significant and justify further clinical evaluation.

Osteogenesis imperfecta (OI) is characterized by osteopenia and bone fragility, and OI patients during growth often exhibit high bone turnover with the net result of low bone mass. Recent evidence shows that osteocytes significantly affect bone remodeling under physiological and pathological conditions through production of osteoclastogenic cytokines. The receptor activator of nuclear factor kappa-B ligand (RANKL) produced by osteocytes for example, is a critical mediator of bone loss caused by ovariectomy, low-calcium diet, unloading and glucocorticoid treatment. Because OI bone has increased density of osteocytes and these cells are embedded in matrix with abnormal type I collagen, we hypothesized that osteocyte-derived RANKL contributes to the OI bone phenotype. In this study, the conditional loss of RANKL in osteocytes in oim/oim mice (oim-RANKL-cKO) resulted in dramatically increased cancellous bone mass in both the femur and lumbar spine compared to oim/oim mice. Bone cortical thickness increased significantly only in spine but ultimate bone strength in the long bone and spine was minimally improved in oim-RANKL-cKO mice compared to oim/oim mice. Furthermore, unlike previous findings, we report that oim/oim mice do not exhibit high bone turnover suggesting that their low bone mass is likely due to defective bone formation and not increased bone resorption. The loss of osteocyte-derived RANKL further diminished parameters of formation in oim-RANKL-cKO. Our results indicate that osteocytes contribute significantly to the low bone mass observed in OI and the effect of loss of RANKL from these cells is similar to its systemic inhibition. Keywords: Osteocyte, RANKL, Osteogenesis imperfecta, Bone fragility

The Leprecan protein family which includes the prolyl 3-hydroxylase enzymes (P3H1, P3H2, and P3H3), the closely related cartilage-associated protein (CRTAP), and SC65 (Synaptonemal complex 65, aka P3H4, LEPREL4), is involved in the post-translational modification of fibrillar collagens. Mutations in CRTAP, P3H1 and P3H2 cause human genetic diseases. We recently showed that SC65 forms a stable complex in the endoplasmic reticulum with P3H3 and lysyl hydroxylase 1 and that loss of this complex leads to defective collagen lysyl hydroxylation and causes low bone mass and skin fragility. Interestingly, SC65 was initially described as a synaptonemal complex-associated protein, suggesting a potential additional role in germline cells. In the present study, we describe the expression of SC65, CRTAP and other Leprecan proteins in postnatal mouse reproductive organs. We detect SC65 expression in peritubular cells of testis up to 4 weeks of age but not in cells within seminiferous tubules, while its expression is maintained in ovarian follicles until adulthood. Similar to bone and skin, SC65 and P3H3 are also tightly co-expressed in testis and ovary. Moreover, we show that CRTAP, a protein normally involved in collagen prolyl 3-hydroxylation, is highly expressed in follicles and stroma of the ovary and in testes interstitial cells at 4 weeks of age, germline cells and mature sperm. Importantly, CrtapKO mice have a mild but significant increase in morphologically abnormal mature sperm (17% increase compared to WT). These data suggest a role for the Leprecans in the post-translational modification of collagens expressed in the stroma of the reproductive organs. While we could not confirm that SC65 is part of the synaptonemal complex, the expression of CRTAP in the seminiferous tubules and in mature sperm suggest a role in the testis germ cell lineage and sperm morphogenesis.

Abstract Osteocytes are long‐lived, highly interconnected, terminally differentiated osteoblasts that reside within mineralized bone matrix. They constitute about 95% of adult bone cells and play important functions including in the regulation of bone remodeling, phosphate homeostasis, and mechanical stimuli sensing and response. However, the role of osteocytes in the pathogenesis of congenital diseases of abnormal bone matrix is poorly understood. This study characterized in vivo transcriptional changes in osteocytes from CrtapKO and oim/oim mouse models of osteogenesis imperfecta (OI) compared with wild‐type (WT) control mice. To do this, RNA was extracted from osteocyte‐enriched cortical femurs and tibias, sequenced and subsequently analyzed to identify differentially expressed transcripts. These models were chosen because they mimic two types of OI with different genetic mutations that result in distinct type I collagen defects. A large number of transcripts were dysregulated in either model of OI, but 281 of them were similarly up‐ or downregulated in both compared with WT controls. Conversely, very few transcripts were differentially expressed between the CrtapKO and oim/oim mice, indicating that distinct alterations in type I collagen can lead to shared pathogenic processes and similar phenotypic outcomes. Bioinformatics analyses identified several critical hubs of dysregulation that were enriched in annotation terms such as development and differentiation, ECM and collagen fibril organization, cell adhesion, signaling, regulatory processes, pattern binding, chemotaxis, and cell projections. The data further indicated alterations in important signaling pathways such as WNT and TGF‐β but also highlighted new candidate genes to pursue in future studies. Overall, our study suggested that the osteocyte transcriptome is broadly dysregulated in OI with potential long‐term consequences at the cellular level, which deserve further investigations. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

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.

Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1(H662A) ). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity versus complete ablation of the prolyl 3-hydroxylation complex.

Mutations in CRTAP (coding for cartilage-associated protein), LEPRE1 (coding for prolyl 3-hydroxylase 1 [P3H1]) or PPIB (coding for Cyclophilin B [CYPB]) cause recessive forms of osteogenesis imperfecta and loss or decrease of type I collagen prolyl 3-hydroxylation. A comprehensive analysis of the phenotype of the Crtap-/- mice revealed multiple abnormalities of connective tissue, including in the lungs, kidneys, and skin, consistent with systemic dysregulation of collagen homeostasis within the extracellular matrix. Both Crtap-/- lung and kidney glomeruli showed increased cellular proliferation. Histologically, the lungs showed increased alveolar spacing, while the kidneys showed evidence of segmental glomerulosclerosis, with abnormal collagen deposition. The Crtap-/- skin had decreased mechanical integrity. In addition to the expected loss of proline 986 3-hydroxylation in alpha1(I) and alpha1(II) chains, there was also loss of 3Hyp at proline 986 in alpha2(V) chains. In contrast, at two of the known 3Hyp sites in alpha1(IV) chains from Crtap-/- kidneys there were normal levels of 3-hydroxylation. On a cellular level, loss of CRTAP in human OI fibroblasts led to a secondary loss of P3H1, and vice versa. These data suggest that both CRTAP and P3H1 are required to maintain a stable complex that 3-hydroxylates canonical proline sites within clade A (types I, II, and V) collagen chains. Loss of this activity leads to a multi-systemic connective tissue disease that affects bone, cartilage, lung, kidney, and skin.