Sex differences in serum phosphate and calcium have been reported but the exact nature and underlying regulatory mechanisms remain unclear. We aimed to compare calcium and phosphate concentrations ...between sexes, and explore potential covariates to elucidate underlying mechanisms of sex differences in a prospective, population-based cohort study. Pooled data of subjects > 45 years from three independent cohorts of the Rotterdam Study (RS) were used: RS-I-3 (n = 3623), RS-II-1 (n = 2394), RS-III-1 (n = 3241), with separate analyses from an additional time point of the first cohort RS-I-1 (n = 2688). Compared to men, women had significantly higher total serum calcium and phosphate concentrations which was not explained by BMI, kidney function nor smoking. Adjustment for serum estradiol diminished sex differences in serum calcium while adjustment for serum testosterone diminished sex differences in serum phosphate. Adjustment for vitamin D and alkaline phosphatase did not change the association between sex and calcium or phosphate in RS-I-1. In the sex-combined group, both serum calcium and phosphate decreased with age with a significant interaction for sex differences for serum calcium but not phosphate. In sex-stratified analyses, serum estradiol but not testosterone was inversely associated with serum calcium in both sexes. Serum estradiol was inversely associated with serum phosphate in both sexes to a similar degree, while serum testosterone was inversely associated with serum phosphate in both sexes with an apparent stronger effect in men than in women. Premenopausal women had lower serum phosphate compared to postmenopausal women. Serum testosterone was inversely associated with serum phosphate in postmenopausal women only. In conclusion, women > 45 years have higher serum calcium and phosphate concentrations compared to men of similar age, not explained by vitamin D or alkaline phosphatase concentrations. Serum estradiol but not testosterone was inversely associated with serum calcium while serum testosterone was inversely associated with serum phosphate in both sexes. Serum testosterone may in part explain sex differences in serum phosphate while estradiol could partly explain sex differences in serum calcium.
Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate ...differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs). Gene expression profiling was performed in hMSCs differentiated toward osteoblasts (at 6 h). Significantly regulated genes were analyzed in silico, and the Connectivity Map (CMap) was used to identify candidate bone stimulatory compounds. The signature of parbendazole matches the expression changes observed for osteogenic hMSCs. Parbendazole stimulates osteoblast differentiation as indicated by increased alkaline phosphatase activity, mineralization, and up-regulation of bone marker genes (alkaline phosphatase/ALPL,osteopontin/SPP1,and bone sialoprotein II/IBSP) in a subset of the hMSC population resistant to the apoptotic effects of parbendazole. These osteogenic effects are independent of glucocorticoids because parbendazole does not up-regulate glucocorticoid receptor (GR) target genes and is not inhibited by the GR antagonist mifepristone. Parbendazole causes profound cytoskeletal changes including degradation of microtubules and increased focal adhesions. Stabilization of microtubules by pretreatment with Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Cotreatment with the BMP-2 antagonist DMH1 limits, but does not block, parbendazole-induced mineralization. Using the CMap we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteogenic differentiation through a combination of cytoskeletal changes and increased BMP-2 activity.
Abstract The response of osteoprogenitors to calcium (Ca2+ ) is of primary interest for both normal bone homeostasis and the clinical field of bone regeneration. The latter makes use of calcium ...phosphate-based bone void fillers to heal bone defects, but it is currently not known how Ca2+ released from these ceramic materials influences cells in situ. Here, we have created an in vitro environment with high extracellular Ca2+ concentration and investigated the response of human bone marrow-derived mesenchymal stromal cells (hMSCs) to it. Ca2+ enhanced proliferation and morphological changes in hMSCs. Moreover, the expression of osteogenic genes is highly increased. A 3-fold up-regulation of BMP-2 is observed after only 6 h and pharmaceutical interference with a number of proteins involved in Ca2+ sensing showed that not the calcium sensing receptor, but rather type L voltage-gated calcium channels are involved in mediating the signaling pathway between extracellular Ca2+ and BMP-2 expression. MEK1/2 activity is essential for the effect of Ca2+ and using microarray analysis, we have identified c-Fos as an early Ca2+ response gene. We have demonstrated that hMSC osteogenesis can be induced via extracellular Ca2+ , a simple and economic way of priming hMSCs for bone tissue engineering applications.
Ectopic vascular calcifications represent a major clinical problem associated with cardiovascular disease and mortality. However, the mechanisms underlying pathological vascular calcifications are ...largely unknown hampering the development of therapies to tackle this life threatening medical condition.
In order to gain insight into the genes and mechanisms driving this pathological calcification process we analyzed the transcriptional profile of calcifying vascular smooth muscle cells (C-VSMCs). These profiles were compared to differentiating osteoblasts, cells that constitute their physiological calcification counterparts in the body. Overall the transcriptional program of C-VSMC and osteoblasts did not overlap. Several genes, some of them relevant for bone formation, were distinctly modulated by C-VSMCs which did not necessarily lose their smooth muscle cell markers while calcifying. Bioinformatics gene clustering and correlation analysis disclosed limited bone-related mechanisms being shared by two cell types. Extracellular matrix (ECM) and biomineralization genes represented common denominators between pathological vascular and physiological bone calcifications. These genes constitute the strongest link between these cells and represent potential drivers for their shared end-point phenotype.
The analyses support the hypothesis that VSMC trans-differentiate into C-VSMCs keeping their own identity while using mechanisms that osteoblasts use to mineralize. The data provide novel insights into groups of genes and biological processes shared in MSC and VSMC osteogenic differentiation. The distinct gene regulation between C-VSMC and osteoblasts might hold clues to find cell-specific pathway modulations, opening the possibility to tackle undesired vascular calcifications without disturbing physiologic bone formation and vice versa.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Bone quality is an important determinant of osteoporosis, and proper osteoblast differentiation plays an important role in the control and maintenance of bone quality. We investigated the impact of ...activin signaling on human osteoblast differentiation, extracellular matrix formation, and mineralization. Activins belong to the transforming growth factor-β superfamily and activin A treatment strongly inhibited mineralization in osteoblast cultures, whereas the activin antagonist follistatin increased mineralization. Osteoblasts produced activin A and follistatin in a differentiation-dependent manner, leading to autocrine regulation of extracellular matrix formation and mineralization. In addition, mineralization in a vascular smooth muscle cell-based model for pathological calcification was inhibited. Comparative activin A and follistatin gene expression profiling showed that activin signaling changes the expression of a specific range of extracellular matrix proteins prior to the onset of mineralization, leading to a matrix composition with reduced or no mineralizing capacity. These findings demonstrate the regulation of osteoblast differentiation and matrix mineralization by the activin A-follistatin system, providing the possibility to control bone quality as well as pathological calcifications such as atherosclerosis by using activin A, follistatin, or analogs thereof.--Eijken, M., Swagemakers, S., Koedam, M., Steenbergen, C., Derkx, P., Uitterlinden, A. G., van der Spek, P. J., Visser, J. A., de Jong, F. H., Pols, H. A. P., van Leeuwen, J. P. T. M. The activin A-follistatin system: potent regulator of human extracellular matrix mineralization.
Arboviruses target bone forming osteoblasts and perturb bone remodeling via paracrine factors. We previously reported that Zika virus (ZIKV) infection of early‐stage human mesenchymal stromal cells ...(MSCs) inhibited the osteogenic lineage commitment of MSCs. To understand the physiological interplay between bone development and ZIKV pathogenesis, we employed a primary in vitro model to examine the biological responses of MSCs to ZIKV infection at different stages of osteogenesis. Precommitted MSCs were infected at the late stage of osteogenic stimulation (Day 7) with ZIKV (multiplicity of infection of 5). We observe that MSCs infected at the late stage of differentiation are highly susceptible to ZIKV infection similar to previous observations with early stage infected MSCs (Day 0). However, in contrast to ZIKV infection at the early stage of differentiation, infection at a later stage significantly elevates the key osteogenic markers and calcium content. Comparative RNA sequencing (RNA‐seq) of early and late stage infected MSCs reveals that ZIKV infection alters the mRNA transcriptome during osteogenic induction of MSCs (1251 genes). ZIKV infection provokes a robust antiviral response at both stages of osteogenic differentiation as reflected by the upregulation of interferon responsive genes (n > 140). ZIKV infection enhances the expression of immune‐related genes in early stage MSCs while increasing cell cycle genes in late stage MSCs. Remarkably, ZIKA infection in early stage MSCs also activates lipid metabolism‐related pathways. In conclusion, ZIKV infection has differentiation stage‐dependent effects on MSCs and this mechanistic understanding may permit the development of new therapeutic or preventative measures for bone‐related effects of ZIKV infection.
For Zika virus (ZIKV), we had previously reported that ZIKV infection of early‐stage human mesenchymal stromal cells (MSCs) inhibited the differentiation and commitment of MSCs. In this follow‐up study, we aimed to determine the effect of ZIKV on late‐staged MSCs. We observed that ZIKV infected the late‐staged MSCs, however, in contrast to ZIKV infection at the early stage of differentiation, infection at a later stage significantly elevated the key osteogenic markers and calcium content. The RNA sequencing data of early and late‐stage infected MSCs also revealed that ZIKV infection altered the mRNA transcriptome during osteogenic induction of MSCs (1251 genes). ZIKV infection enhanced the expression of immune‐related genes in early‐stage MSCs while increasing cell cycle genes in late‐stage MSCs specifically. In conclusion, we observed the differentiation stage‐dependent effects of ZIKV infection, and this mechanistic understanding may permit the development of new therapeutic or preventative measures for bone‐related effects of ZIKV infection.
ABSTRACTInhibitors of the activin receptor signaling pathway (IASPs) have become candidate therapeutics for sarcopenia and bone remodeling disorders because of their ability to increase muscle and ...bone mass. However, IASPs utilizing activin type IIA and IIB receptors are also potent stimulators of erythropoiesis, a feature that may restrict their usage to anemic patients because of increased risk of venous thromboembolism. Based on the endogenous TGF‐β superfamily antagonist follistatin (FST), a molecule in the IASP class, FSTΔHBS‐mFc, was generated and tested in both ovariectomized and naive BALB/c and C57BL/6 mice. In ovariectomized mice, FSTΔHBS‐Fc therapy dose‐dependently increased cancellous bone mass up to 42% and improved bone microstructural indices. For the highest dosage of FSTΔHBS‐mFc (30 mg/kg, 2 times/wk), the increase in cancellous bone mass was similar to that observed with parathyroid hormone therapy (1–34,80 µg/kg, 5 times/wk). Musculus quadriceps femoris mass dose‐dependently increased up to 21% in ovariectomized mice. In both ovariectomized and naive mice, FSTΔHBS‐mFc therapy did not influence red blood cell count or hematocrit or hemoglobin levels. If the results are reproduced, a human FSTΔHBS‐mFc version could be applicable in patients with musculoskeletal conditions irrespective of hematocrit status.—Lodberg, A., van der Eerden, B. C. J., Boers‐Sijmons, B., Thomsen, J. S., Brüel, A., van Leeuwen, J. P. T. M., Eijken, M. A follistatin‐based molecule increases muscle and bone mass without affecting the red blood cell count in mice. FASEB J. 33, 6001–6010 (2019). www.fasebj.org
Bone marrow derived mesenchymal stromal cells (BMSCs) are multipotent progenitors of particular interest for cell-based tissue engineering therapies. However, one disadvantage that limit their ...clinical use is their heterogeneity. In the last decades a great effort was made to select BMSC subpopulations based on cell surface markers, however there is still no general consensus on which markers to use to obtain the best BMSCs for tissue regeneration. Looking for alternatives we decided to focus on a probe-based method to detect intracellular mRNA in living cells, the SmartFlare technology. This technology does not require fixation of the cells and allows us to sort living cells based on gene expression into functionally different populations. However, since the technology is available it is debated whether the probes specifically recognize their target mRNAs. We validated the TWIST1 probe and demonstrated that it specifically recognizes TWIST1 in BMSCs. However, differences in probe concentration, incubation time and cellular uptake can strongly influence signal specificity. In addition we found that
TWIST1
high
expressing cells have an increased expansion rate compared to
TWIST1
low
expressing cells derived from the same initial population of BMSCs. The SmartFlare probes recognize their target gene, however for each probe and cell type validation of the protocol is necessary.
Bone is a dynamic tissue that is strongly influenced by endocrine factors to restore the balance between bone resorption and bone formation. Bone formation involves the mineralization of the ...extracellular matrix formed by osteoblasts. In this process the role of vitamin D (1α,25(OH)2D3) is both direct and indirect. The direct effects are enabled via the Vitamin D Receptor (VDR); the outcome is dependent on the presence of other factors as well as origin of the osteoblasts, treatment procedures and species differences. Vitamin D stimulates mineralization of human osteoblasts but is often found inhibitory for mineralization of murine osteoblasts.
In this review we will overview the current knowledge of the role of the vitamin D endocrine system in controlling the mineralization process in bone.
•Vitamin D is important for mineralization of bone tissue, either direct or indirect.•The role of vitamin D in bone mineralization is species and osteoblast differentiation stage dependent.•Vitamin D actions on bone mineralization are part of many networks.
Activins regulate bone formation by controlling osteoclasts and osteoblasts. We investigated Activin‐A mechanism of action on human osteoblast mineralization, RNA and microRNA (miRNA) expression ...profile. A single 2‐day treatment of Activin‐A at Day 5 of osteoblast differentiation significantly reduced matrix mineralization. Activin A‐treated osteoblasts responded with transient change in gene expression, in a 2‐wave‐fashion. The 38 genes differentially regulated during the first wave (within 8 hr after Activin A start) were involved in transcription regulation. In the second wave (1–2 days after Activin A start), 65 genes were differentially regulated and related to extracellular matrix. Differentially expressed genes in both waves were associated to transforming growth factor beta signaling. We identified which microRNAs modulating osteoblast differentiation were regulated by Activin‐A. In summary, 2‐day treatment with Activin‐A in premineralization period of osteoblast cultures influenced miRNAs, gene transcription, and reduced matrix mineralization. Modulation of Activin A signaling might be useful to control bone quality for therapeutic purposes.
A single 2‐day treatment of Activin‐A in the premineralization period of osteoblast cultures significantly reduced extracellular matrix mineralization 5–7 days later.
Activin‐A treatment induced a transient change in osteoblast gene expression in a 2‐wave‐fashion over time. Differentially regulated genes were involved in transcription regulation, extracellular matrix structure, and associated to transforming growth factor beta signaling. Activin‐A modulated osteoblast microRNA profiles.
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