<i>In</i> <i>vitro </i>regulation of fibroblast growth factor 23 by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D synthesized by osteocyte-like MC3T3-E1 cells

Fibroblast growth factor 23 (FGF23) is produced and secreted by osteocytes and is essential for maintaining phosphate homeostasis. One of the main regulators of FGF23, 1,25-dihydroxyvitamin D (1,25(OH)2D3), is primarily synthesized in the kidney from 25-hydroxyvitamin D (25(OH)D) by 1α-hydroxylase (encoded by CYP27B1). Hitherto, it is unclear whether osteocytes can convert 25(OH)D and thereby allow for 1,25(OH)2D3 to induce FGF23 production and secretion locally. Here, we differentiated MC3T3-E1 cells toward osteocyte-like cells expressing and secreting FGF23. Treatment with 10-6 M 25(OH)D resulted in conversion of 25(OH)D to 150 pmol/L 1,25(OH)2D3 and increased FGF23 expression and secretion, but the converted amount of 1,25(OH)2D3 was insufficient to trigger an FGF23 response, so the effect on FGF23 was most likely directly caused by 25(OH)D. Interestingly, combining phosphate with 25(OH)D resulted in a synergistic increase in FGF23 expression and secretion, likely due to activation of additional signaling pathways by phosphate. Blockage of the vitamin D receptor (VDR) only partially abolished the effects of 25(OH)D or 25(OH)D combined with phosphate on Fgf23, while completely inhibiting the upregulation of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1), encoding for 24-hydroxylase. RNA sequencing and in silico analyses showed that this could potentially be mediated by the nuclear receptors Retinoic Acid Receptor β (RARB) and Estrogen Receptor 2 (ESR2). Taken together, we demonstrate that osteocytes are able to convert 25(OH)D to 1,25(OH)2D3, but this is insufficient for FGF23 activation, implicating a direct effect of 25(OH)D in the regulation of FGF23, which occurs at least partially independent from its cognate VDR. Moreover, phosphate and 25(OH)D synergistically increase expression and secretion of FGF23, which warrants investigating consequences in patients receiving a combination of vitamin D analogues and phosphate supplements. These observations help us to further understand the complex relations between phosphate, vitamin D, and FGF23.</p

Tensin-3 is involved in osteogenic versus adipogenic fate of human bone marrow stromal cells

Background: The tightly controlled balance between osteogenic and adipogenic differentiation of human bone marrow-derived stromal cells (BMSCs) is critical to maintain bone homeostasis. Age-related osteoporosis is characterized by low bone mass with excessive infiltration of adipose tissue in the bone marrow compartment. The shift of BMSC differentiation from osteoblasts to adipocytes could result in bone loss and adiposity. Methods: TNS3 gene expression during osteogenic and adipogenic differentiation of BMSCs was evaluated by qPCR and Western blot analyses. Lentiviral-mediated knockdown or overexpression of TNS3 was used to assess its function. The organization of cytoskeleton was examined by immunofluorescent staining at multiple time points. The role of TNS3 and its domain function in osteogenic differentiation were evaluated by ALP activity, calcium assay, and Alizarin Red S staining. The expression of Rho-GTP was determined using the RhoA pull-down activation assay. Results:Loss of TNS3 impaired osteogenic differentiation of BMSCs but promoted adipogenic differentiation. Conversely, TNS3 overexpression hampered adipogenesis while enhancing osteogenesis. The expression level of TNS3 determined cell shape and cytoskeletal reorganization during osteogenic differentiation. TNS3 truncation experiments revealed that for optimal osteogenesis to occur, all domains proved essential. Pull-down and immunocytochemical experiments suggested that TNS3 mediates osteogenic differentiation through RhoA. Conclusions: Here, we identify TNS3 to be involved in BMSC fate decision. Our study links the domain structure in TNS3 to RhoA activity via actin dynamics and implicates an important role for TNS3 in regulating osteogenesis and adipogenesis from BMSCs. Furthermore, it supports the critical involvement of cytoskeletal reorganization in BMSC differentiation.</p

Organic phosphate but not inorganic phosphate regulates Fgf23 expression through MAPK and TGF-ꞵ signaling

One of the main regulators of phosphate homeostasis is fibroblast growth factor 23 (FGF23), secreted by osteocytes. The effects of organic versus inorganic dietary phosphate on this homeostasis are unclear. This study used MC3T3-E1 FGF23-producing cells to examine the transcriptomic responses to these phosphates. Most importantly, the expression and secretion of FGF23 were only increased in response to organic phosphate. Gene ontology terms related to a response to environmental change were only enriched in cells treated with organic phosphate while cells treated with inorganic phosphate were enriched for terms associated with regulation of cellular phosphate metabolism. Inhibition of MAPK signaling diminished the response of Fgf23 to organic phosphate, suggesting it activates FGF23. TGF-β signaling inhibition increased Fgf23 expression after the addition of organic phosphate, while the negative TGF-β regulator Skil decreased this response. In summary, the observed differential response of FGF23-producing to phosphate types may have consequences for phosphate homeostasis.</p

Organic phosphate but not inorganic phosphate regulates Fgf23 expression through MAPK and TGF-ꞵ signaling

One of the main regulators of phosphate homeostasis is fibroblast growth factor 23 (FGF23), secreted by osteocytes. The effects of organic versus inorganic dietary phosphate on this homeostasis are unclear. This study used MC3T3-E1 FGF23-producing cells to examine the transcriptomic responses to these phosphates. Most importantly, the expression and secretion of FGF23 were only increased in response to organic phosphate. Gene ontology terms related to a response to environmental change were only enriched in cells treated with organic phosphate while cells treated with inorganic phosphate were enriched for terms associated with regulation of cellular phosphate metabolism. Inhibition of MAPK signaling diminished the response of Fgf23 to organic phosphate, suggesting it activates FGF23. TGF-β signaling inhibition increased Fgf23 expression after the addition of organic phosphate, while the negative TGF-β regulator Skil decreased this response. In summary, the observed differential response of FGF23-producing to phosphate types may have consequences for phosphate homeostasis.</p

Hydroxychloroquine decreases human MSC-derived osteoblast differentiation and mineralization in vitro

We recently showed that patients with primary Sjögren Syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favourable effects on BMD. To study the direct effects of HCQ on human MSC-derived osteoblast activity. Osteoblasts were cultured from human mesenchymal stromal cells (hMSCs). Cultures were treated with different HCQ doses (control, 1 and 5 µg/ml). Alkaline phosphatase activity and calcium measurements were performed to evaluate osteoblast differentiation and activity, respectively. Detailed microarray analysis was performed in 5 µg/ml HCQ-treated cells and controls followed by qPCR validation. Additional cultures were performed using the cholesterol synthesis inhibitor simvastatin (SIM) to evaluate a potential mechanism of action. We showed that HCQ inhibits both MSC-derived osteoblast differentiation and mineralization in vitro. Microarray analysis and additional PCR validation reveale

Identification of small molecules as novel anti-adipogenic compounds based on Connectivity Map

Several physiological and pathological conditions such as aging, obesity, diabetes, anorexia nervosa are associated with increased adipogenesis in the bone marrow. A lack of effective drugs hinder the improved treatment for aberrant accumulation of bone marrow adipocytes. Given the higher costs, longer duration and sometimes lack of efficacy in drug discovery, computational and experimental strategies have been used to identify previously approved drugs for the treatment of diseases, also known as drug repurposing. Here, we describe the method of small molecule-prioritization by employing adipocyte-specific genes using the connectivity map (CMap). We then generated transcriptomic profiles using human mesenchymal stromal cells under adipogenic differentiation with the treatment of prioritized compounds, and identified emetine and kinetin-riboside to have a potent inhibitory effect on adipogenesis. Overall, we demonstrated a proof-of-concept method to identify repurposable drugs capable of inhibiting adipogenesis, using the Connectivity Map

Investigating the role of ASCC1 in the causation of bone fragility

Bi-allelic variants in ASCC1 cause the ultrarare bone fragility disorder “spinal muscular atrophy with congenital bone fractures-2” (SMABF2). However, the mechanism by which ASCC1 dysfunction leads to this musculoskeletal condition and the nature of the associated bone defect are poorly understood. By exome sequencing, we identified a novel homozygous deletion in ASCC1 in a female infant. She was born with severe muscular hypotonia, inability to breathe and swallow, and virtual absence of spontaneous movements; showed progressive brain atrophy, gracile long bones, very slender ribs, and a femur fracture; and died from respiratory failure aged 3 months. A transiliac bone sample taken postmortem revealed a distinct microstructural bone phenotype with low trabecular bone volume, low bone remodeling, disordered collagen organization, and an abnormally high bone marrow adiposity. Proteomics, RNA sequencing, and qPCR in patient-derived skin fibroblasts confirmed that ASCC1 was hardly expressed on protein and RNA levels compared with healthy controls. Furthermore, we demonstrate that mutated ASCC1 is associated with a downregulation of RUNX2, the master regulator of osteoblastogenesis, and SERPINF1, which is involved in osteoblast and adipocyte differentiation. It also exerts an inhibitory effect on TGF-β/SMAD signaling, which is important for bone development. Additionally, knockdown of ASCC1 in human mesenchymal stromal cells (hMSCs) suppressed their differentiation capacity into osteoblasts while increasing their differentiation into adipocytes. This resulted in reduced mineralization and elevated formation of lipid droplets. These findings shed light onto the pathophysiologic mechanisms underlying SMABF2 and assign a new biological role to ASCC1 acting as an important pro-osteoblastogenic and anti-adipogenic regulator.</p