Mammalian high-temperature requirement serine protease A1 (HTRA1) is a secreted member of the trypsin family of serine proteases which can degrade a variety of bone matrix proteins and as such has been implicated in musculoskeletal development. In this study, we have investigated the role of HTRA1 in mesenchymal stem cell (MSC) osteogenesis and suggest a potential mechanism through which it controls matrix mineralization by differentiating boneforming cells. Osteogenic induction resulted in a significant elevation in the expression and secretion of HTRA1 in MSCs isolated from human bone marrow-derived MSCs (hBMSCs), mouse adipose-derived stromal cells (mASCs), and mouse embryonic stem cells. Recombinant HTRA1 enhanced the osteogenesis of hBMSCs as evidenced by significant changes in several osteogenic markers including integrin-binding sialoprotein (IBSP), bone morphogenetic protein 5 (BMP5), and sclerostin, and promoted matrix mineralization in differentiating boneforming osteoblasts. These stimulatory effects were not observed with proteolytically inactive HTRA1 and were abolished by small interfering RNA against HTRA1. Moreover, loss of HTRA1 function resulted in enhanced adipogenesis of hBMSCs. HTRA1 Immunofluorescence studies showed colocalization of HTRA1 with IBSP protein in osteogenic mASC spheroid cultures and was confirmed as being a newly identified HTRA1 substrate in cell cultures and in proteolytic enzyme assays. A role for HTRA1 in bone regeneration in vivo was also alluded to in bone fracture repair studies where HTRA1 was found localized predominantly to areas of new bone formation in association with IBSP. These data therefore implicate HTRA1 as having a central role in osteogenesis through modification of proteins within the extracellular matrix. STEM CELLS

Adipose tissue provides for a rich and easily accessible source of multipotent stromal cells and thus offers the potential for autologous cell-based therapy for a number of degenerative diseases. Senile osteoporosis is characterized by a reduction in bone quality, which is associated with inadequacies in bone marrow stromal cell (BMSC) differentiation. In the present study, we have characterized adipose-derived stromal cells (ASCs) isolated from aged osteoporotic mice and evaluated their suitability as a source of osteogenic precursor cells. Significant reductions in both tibia bone quality and telomere length in liver tissue were observed in the senescence-accelerated mouse prone 6 strain (SAMP6), as compared to the control age-matched senescence-accelerated mouse resistant 1 strain (SAMR1), thus confirming osteoporosis and accelerated ageing traits in this model. ASCs isolated from inguinal fat expressed mesenchymal surface markers and were capable of differentiating along the osteoblast, adipocyte and chondrocyte lineages. Telomere length was not compromised in ASCs from SAMP6 mice but was actually found to be significantly increased as compared to control SAMR1 mice. Furthermore, ASCs from both strains were comparable in terms of telomerase activity, p21 mRNA expression, SA-β-gal activity and proliferative capacity. The overall osteogenic and adipogenic potential of ASCs was comparable between SAMP6 and SAMR1 strains, as determined by quantitative molecular, biochemical and histological analyses. In conclusion, adipose tissue may represent a promising autologous cell source for the development of novel bone regenerative therapeutic strategies in the treatment of age-related osteoporosis.

Background: HTRA1 has been associated with intervertebral disc (IVD) degeneration although its role is unknown. Results: HTRA1 up-regulated matrix metalloproteinase (MMP) production by IVD cells via the generation of fibronectin fragments. Conclusion: HTRA1 plays a detrimental role in the pathogenesis of IVD degeneration. Significance: HTRA1 may represent a novel therapeutic target for the treatment of spinal disc degeneration.

ADP-ribosyltransferase Diphtheria toxin-like 1 [ARTD1; formerly called poly-ADP-ribose polymerase 1 (PARP1)] is a chromatin-associated enzyme involved in regulating metabolic homeostasis. The liver is at the core of glucose and lipid metabolism and is significantly affected by obesity and the metabolic syndrome. Here, we show that when fed a high-fat diet (HFD), mice lacking ARTD1 developed exacerbated hepatic steatosis. ARTD1(-/-) mice had a 19% higher liver weight than wild-type (WT) animals and exhibited a significantly increased serum concentration of cholesterol (38%) and impaired glucose tolerance. In addition, adipocyte function and size were significantly reduced in ARTD1(-/-) mice fed an HFD (7794 μm(2) for WT and 5579 μm(2) for ARTD1(-/-) mice). The significantly reduced adipogenic differentiation of adipose-derived stromal cells (ASCs) isolated from ARTD1(-/-) mice (28 vs. 11% Oil red O-positive cells in WT and ARTD1(-/-) ASCs, respectively) suggested that impaired adipogenesis as the underlying cause for this adipose tissue malfunction. This function of ARTD1 was specific for adipogenesis, since osteogenic differentiation was not affected by the ARTD1 deletion. In summary, we show that ARTD1(-/-) mice fed an HFD display impaired adipogenesis and show exacerbated hepatic steatosis, which can have important implications for nonalcoholic fatty liver disease.

a b s t r a c tAdipose-derived stromal cells (ASCs) are increasingly being used for orthopedic-based tissue engineering approaches due to their ability to readily undergo osteogenic differentiation. In the present study, we used in vitro and in vivo approaches to evaluate the use of ASCs as a treatment strategy for age-related osteoporosis. Molecular, histological and micro-computed tomography (micro-CT) based approaches confirmed that ASCs isolated from 18-week-old osteoporotic senescence-accelerated mice (SAMP6) were capable of undergoing osteogenesis when cultured in either silk fibroin (SF) scaffolds or scaffold-free microtissues (ASC-MT). A single intratibial injection of CM-Dil-labeled isogeneic ASCs or ASC-MT into SAMP6 recipients significantly improved trabecular bone quality after 6 weeks in comparison to untreated contralateral bones, as determined by micro-CT. Injected ASCs could be observed in paraffin wax bone sections at 24 h and 6 weeks post treatment and induced a significant increase in several molecular markers of bone turnover. Furthermore, a significant improvement in the osteogenic potential of osteoporotic patient-derived human bone marrow stromal cells (BMSCs) was observed when differentiated in conditioned culture media harvested from osteoporotic patient-derived human ASCs. These findings therefore support the use of ASCs as an autologous cell-based approach for the treatment of osteoporosis.

PPARγ-dependent gene expression during adipogenesis is facilitated by ADP-ribosyltransferase D-type 1 (ARTD1; PARP1)-catalyzed poly-ADP-ribose (PAR) formation. Adipogenesis is accompanied by a dynamic modulation of the chromatin landscape at PPARγ target genes by ligand-dependent co-factor exchange. However, how endogenous PPARγ ligands, which have a low affinity for the receptor and are present at low levels in the cell, can induce sufficient co-factor exchange is unknown. Moreover, the significance of PAR formation in PPARγ-regulated adipose tissue function is also unknown. Here, we show that inhibition of PAR formation in mice on a high-fat diet reduces weight gain and cell size of adipocytes, as well as PPARγ target gene expression in white adipose tissue. Mechanistically, topoisomerase II activity induces ARTD1 recruitment to PPARγ target genes, and ARTD1 automodification enhances ligand binding to PPARγ, thus promoting sufficient transcriptional co-factor exchange in adipocytes. Thus, ARTD1-mediated PAR formation during adipogenesis is necessary to adequately convey the low signal of endogenous PPARγ ligand to effective gene expression. These results uncover a new regulatory mechanism of ARTD1-induced ADP-ribosylation and highlight its importance for nuclear factor-regulated gene expression.

No abstract

While ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) and its enzymatic activity have been shown to be important for reprogramming and differentiation of cells, such as during adipogenesis, their role and mechanism in regulating osteoclastogenesis and bone homeostasis are largely unknown. Here, in cell culture-based RANKL-induced osteoclastogenesis models, we show that silencing of ARTD1 or inhibition of its enzymatic activity enhances osteoclast differentiation and function. As a consequence of ARTD1 silencing or inhibition, the recruitment of p65/RelA to the IL-1β promoter, which is associated with transcriptionally active histone marks, IL-1β expression and inflammasome-dependent secretion of IL-1β are enhanced. This subsequently promotes sustained induction of the transcription factor Nfatc1/A and osteoclastogenesis in an autocrine manner via the IL-1 receptor. In vivo, Artd1-deficient mice display significantly decreased bone mass as a consequence of increased osteoclast differentiation. Accordingly, the expression of osteoclast markers is enhanced in mutant compared to wild-type mice. Together, these results indicate that ARTD1 controls osteoclast development and bone remodelling via its enzymatic activity by modulating the epigenetic marks surrounding the IL-1β promoter and expression of IL-1β and subsequently also Nfatc1/A.