Terry Bertin

CRTAP Is Required for Prolyl 3- Hydroxylation and Mutations Cause Recessive Osteogenesis Imperfecta

Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.

Dimorphic effects of Notch signaling in bone homeostasis

Notch signaling is a key mechanism in the control of embryogenesis. However, its in vivo function during mesenchymal cell differentiation, and, specifically, in bone homeostasis, remains largely unknown. Here, we show that osteoblast-specific gain of Notch function causes severe osteosclerosis owing to increased proliferation of immature osteoblasts. Under these pathological conditions, Notch stimulates early osteoblastic proliferation by upregulating the genes encoding cyclin D, cyclin E and Sp7 (osterix). The intracellular domain of Notch1 also regulates terminal osteoblastic differentiation by directly binding Runx2 and repressing its transactivation function. In contrast, loss of all Notch signaling in osteoblasts, generated by deletion of the genes encoding presenilin-1 and presenilin-2 in bone, is associated with late-onset, age-related osteoporosis, which in turn results from increased osteoblast-dependent osteoclastic activity due to decreased osteoprotegerin mRNA expression in these cells. Together, these findings highlight the potential dimorphic effects of Notch signaling in bone homeostasis and may provide direction for novel therapeutic applications.

CRTAP AND LEPRE1 MUTATIONS IN RECESSIVE OSTEOGENESIS IMPERFECTA

Autosomal dominant osteogenesis imperfecta (OI) is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Recently, dysregulation of hydroxylation of a single proline residue at position 986 of both the triple‐helical domains of type I collagen α1(I) and type II collagen α1(II) chains has been implicated in the pathogenesis of recessive forms of OI. Two proteins, cartilage‐associated protein (CRTAP) and prolyl‐3‐hydroxylase‐1 (P3H1, encoded by the LEPRE1 gene) form a complex that performs the hydroxylation and brings the prolyl cis‐trans isomerase cyclophilin‐B (CYPB) to the unfolded collagen. In our screen of 78 subjects diagnosed with OI type II or III, we identified three probands with mutations in CRTAP and 16 with mutations in LEPRE1. The latter group includes a mutation in patients from the Irish Traveller population, a genetically isolated community with increased incidence of OI. The clinical features resulting from CRTAP or LEPRE1 loss of function mutations were difficult to distinguish at birth. Infants in both groups had multiple fractures, decreased bone modeling (affecting especially the femurs), and extremely low bone mineral density. Interestingly, “popcorn” epiphyses may reflect underlying cartilaginous and bone dysplasia in this form of OI. These results expand the range of CRTAP/LEPRE1 mutations that result in recessive OI and emphasize the importance of distinguishing recurrence of severe OI of recessive inheritance from those that result from parental germline mosaicism for COL1A1 or COL1A2 mutations. Hum Mutat 0, 1–8, 2008.

miRNA-34c regulates Notch signaling during bone development

During bone homeostasis, osteoblast and osteoclast differentiation is coupled and regulated by multiple signaling pathways and their downstream transcription factors. Here, we show that microRNA 34 (miR-34) is significantly induced by BMP2 during osteoblast differentiation. In vivo, osteoblast-specific gain of miR-34c in mice leads to an age-dependent osteoporosis due to the defective mineralization and proliferation of osteoblasts and increased osteoclastogenesis. In osteoblasts, miR-34c targets multiple components of the Notch signaling pathway, including Notch1, Notch2 and Jag1 in a direct manner, and influences osteoclast differentiation in a non-cell-autonomous fashion. Taken together, our results demonstrate that miR-34c is critical during osteoblastogenesis in part by regulating Notch signaling in bone homeostasis. Furthermore, miR-34c-mediated post-transcriptional regulation of Notch signaling in osteoblasts is one possible mechanism to modulate the proliferative effect of Notch in the committed osteoblast progenitors which may be important in the pathogenesis of osteosarcomas. Therefore, understanding the functional interaction of miR-34 and Notch signaling in normal bone development and in bone cancer could potentially lead to therapies modulating miR-34 signaling.

Mutations in SERPINF1 cause osteogenesis imperfecta type VI

Osteogenesis imperfecta (OI) is a spectrum of genetic disorders characterized by bone fragility. It is caused by dominant mutations affecting the synthesis and/or structure of type I procollagen or by recessively inherited mutations in genes responsible for the posttranslational processing/trafficking of type I procollagen. Recessive OI type VI is unique among OI types in that it is characterized by an increased amount of unmineralized osteoid, thereby suggesting a distinct disease mechanism. In a large consanguineous family with OI type VI, we performed homozygosity mapping and next‐generation sequencing of the candidate gene region to isolate and identify the causative gene. We describe loss of function mutations in serpin peptidase inhibitor, clade F, member 1 (SERPINF1) in two affected members of this family and in an additional unrelated patient with OI type VI. SERPINF1 encodes pigment epithelium–derived factor. Hence, loss of pigment epithelium–derived factor function constitutes a novel mechanism for OI and shows its involvement in bone mineralization.

Notch signaling contributes to the pathogenesis of human osteosarcomas

Notch signaling plays an important role in developmental processes and adult tissue homeostasis. Altered Notch signaling has been associated with various diseases including cancer. While the importance of altered Notch signaling in cancers of hematopoietic and epithelial origins has been established, its role in tumors of mesenchymal origin is less clear. Here, we report that human osteosarcoma cell lines and primary human osteosarcoma tumor samples show significant up-regulation of Notch, its target genes and Osterix. Notch inhibition by gamma-secretase inhibitors or by using lentiviral mediated expression of dominant negative Mastermind-like protein (DN-MAML) decreases osteosarcoma cell proliferation in vitro. In vivo, established human tumor xenografts in nude mice show decreased tumor growth after chemical or genetic inhibition of Notch signaling. Finally, transcriptional profiling of osteosarcomas from p53 mutant mice confirmed up-regulation of Notch1 target genes Hes1, Hey1 and its ligand Dll4. Our data suggest that activation of Notch signaling contributes to the pathogenesis of human osteosarcomas and its inhibition may be a therapeutic approach for the treatment of this mesenchymal tumor.

Requirement of argininosuccinate lyase for systemic nitric oxide production

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases.

Excessive transforming growth factor-β signaling is a common mechanism in osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a heritable disorder, in both a dominant and recessive manner, of connective tissue characterized by brittle bones, fractures and extraskeletal manifestations. How structural mutations of type I collagen (dominant OI) or of its post-translational modification machinery (recessive OI) can cause abnormal quality and quantity of bone is poorly understood. Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms. Here, we show that excessive transforming growth factor-β (TGF-β) signaling is a mechanism of OI in both recessive (Crtap−/−) and dominant (Col1a2tm1.1Mcbr) OI mouse models. In the skeleton, we find higher expression of TGF-β target genes, higher ratio of phosphorylated Smad2 to total Smad2 protein and higher in vivo Smad2 reporter activity. Moreover, the type I collagen of Crtap−/− mice shows reduced binding to the small leucine-rich proteoglycan decorin, a known regulator of TGF-β activity. Anti–TGF-β treatment using the neutralizing antibody 1D11 corrects the bone phenotype in both forms of OI and improves the lung abnormalities in Crtap−/− mice. Hence, altered TGF-β matrix-cell signaling is a primary mechanism in the pathogenesis of OI and could be a promising target for the treatment of OI.

Interaction of TGFβ and BMP Signaling Pathways during Chondrogenesis

TGFβ and BMP signaling pathways exhibit antagonistic activities during the development of many tissues. Although the crosstalk between BMP and TGFβ signaling pathways is well established in bone development, the relationship between these two pathways is less well defined during cartilage development and postnatal homeostasis. We generated hypomorphic mouse models of cartilage-specific loss of BMP and TGFβ signaling to assess the interaction of these pathways in postnatal growth plate homeostasis. We further used the chondrogenic ATDC5 cell line to test effects of BMP and TGFβ signaling on each others downstream targets. We found that conditional deletion of Smad1 in chondrocytes resulted in a shortening of the growth plate. The addition of Smad5 haploinsufficiency led to a more severe phenotype with shorter prehypertrophic and hypertrophic zones and decreased chondrocyte proliferation. The opposite growth plate phenotype was observed in a transgenic mouse model of decreased chondrocytic TGFβ signaling that was generated by expressing a dominant negative form of the TGFβ receptor I (ΔTβRI) in cartilage. Histological analysis demonstrated elongated growth plates with enhanced Ihh expression, as well as an increased proliferation rate with altered production of extracellular matrix components. In contrast, in chondrogenic ATDC5 cells, TGFβ was able to enhance BMP signaling, while BMP2 significantly reduces levels of TGF signaling. In summary, our data demonstrate that during endochondral ossification, BMP and TGFβ signaling can have antagonistic effects on chondrocyte proliferation and differentiation in vivo. We also found evidence of direct interaction between the two signaling pathways in a cell model of chondrogenesis in vitro.

Proteoglycan 4 expression protects against the development of osteoarthritis.

Proteoglycan 4 protects from age-related and posttraumatic osteoarthritis development. Rubbing Arthritis the Wrong Way There are two main forms of joint inflammation (arthritis). Patients with rheumatoid arthritis develop joint inflammation because of an autoimmune reaction, whereas wear and tear is thought to contribute to the development of osteoarthritis. Indeed, most people have at least some symptoms of osteoarthritis by the age of 70. Treatment is mostly limited to reducing symptoms. Now, Ruan et al. provide a new mechanism for a potential new therapy for osteoarthritis. Proteoglycan 4 (lubrican, PRG4) has been previously thought to protect against osteoarthritis by acting as a lubricant between bones in a joint. Here, the authors show that not only does PRG4 protect against the development of osteoarthritis in both aging and trauma mouse models, but that its effect may extend beyond that of a mere lubricant. Indeed, PRG4 has a direct chondroprotective effect in these models, suggesting that it may form the basis for a new disease-altering therapy for osteoarthritis. Osteoarthritis (OA) is a common degenerative condition that afflicts more than 70% of the population between 55 and 77 years of age. Although its prevalence is rising globally with aging of the population, current therapy is limited to symptomatic relief and, in severe cases, joint replacement surgery. We report that intra-articular expression of proteoglycan 4 (Prg4) in mice protects against development of OA. Long-term Prg4 expression under the type II collagen promoter (Col2a1) does not adversely affect skeletal development but protects from developing signs of age-related OA. The protective effect is also shown in a model of posttraumatic OA created by cruciate ligament transection. Moreover, intra-articular injection of helper-dependent adenoviral vector expressing Prg4 protected against the development of posttraumatic OA when administered either before or after injury. Gene expression profiling of mouse articular cartilage and in vitro cell studies show that Prg4 expression inhibits the transcriptional programs that promote cartilage catabolism and hypertrophy through the up-regulation of hypoxia-inducible factor 3α. Analyses of available human OA data sets are consistent with the predictions of this model. Hence, our data provide insight into the mechanisms for OA development and offer a potential chondroprotective approach to its treatment.