Protein arginine methyltransferases PRMT1, PRMT4/CARM1 and PRMT5 have distinct functions in control of osteoblast differentiation

Osteogenic differentiation of mesenchymal cells is controlled by epigenetic enzymes that regulate post-translational modifications of histones. Compared to acetyl or methyltransferases, the physiological functions of protein arginine methyltransferases (PRMTs) in osteoblast differentiation remain minimally understood. Therefore, we surveyed the expression and function of all nine mammalian PRMT members during osteoblast differentiation. RNA-seq gene expression profiling shows that Prmt1, Prmt4/Carm1 and Prmt5 represent the most prominently expressed PRMT subtypes in mouse calvarial bone and MC3T3 osteoblasts as well as human musculoskeletal tissues and mesenchymal stromal cells (MSCs). Based on effects of siRNA depletion, it appears that PRMT members have different functional effects: (i) loss of Prmt1 stimulates and (ii) loss of Prmt5 decreases calcium deposition of mouse MC3T3 osteoblasts, while (iii) loss of Carm1 is inconsequential for calcium deposition. Decreased Prmt5 suppresses expression of multiple genes involved in mineralization (e.g., Alpl, Ibsp, Phospho1) consistent with a positive role in osteogenesis. Depletion of Prmt1, Carm1 and Prmt5 has intricate but modest time-dependent effects on the expression of a panel of osteoblast differentiation and proliferation markers but does not change mRNA levels for select epigenetic regulators (e.g., Ezh1, Ezh2, Brd2 and Brd4). Treatment with the Class I PRMT inhibitor GSK715 enhances extracellular matrix mineralization of MC3T3 cells, while blocking formation of H3R17me2a but not H4R3me2a marks. In sum, Prmt1, Carm1 and Prmt5 have distinct biological roles during osteoblast differentiation, and different types histone H3 and H4 arginine methylation may contribute to the chromatin landscape during osteoblast differentiation.</p

Cytopenia occurrence in kidney transplant recipients within early post-transplant period

Objective: This study assessed incidence, severity, and time to occurrence of drug-induced leukopenia/thrombocytopenia within 1st month after kidney transplantation. Methods: This cross-sectional study was conducted on newly kidney transplant recipients from two hospitals, Iran. Patients with thrombocytopenia due to acute antibody-mediated rejection were excluded from the study. Demographic, clinical, and laboratory data of patients within the 1st month after transplantation were collected. Findings: Of 213 patients, 14.1% and 66.2% experienced leukopenia and thrombocytopenia, respectively. Cytopenia happened more commonly among patients with thymoglobulin-containing regimen (for leukopenia: 24.6% vs. 0%, P < 0.001; for thrombocytopenia 84.4% vs. 41.8%, P < 0.001). Most leukopenia patients experienced Grades 1 and 2 of leukopenia (46.6% and 40% of patients). Most thrombocytopenic patients showed Grade 1 of thrombocytopenia (78.7%). Cumulative dose of thymoglobulin did not differ between patients with and without leukopenia (5.57 ± 1.13 vs. 5.9 ± 1.96 mg/kg; P = 0.613) or with and without thrombocytopenia (5.87 ± 1.86 vs. 5.56 ± 1.38 mg/kg; P = 0.29). Cytopenia were more common among recipients from deceased compared with from living donors (91.3% vs. 8.7% for leukopenia patients, P = 0.001; 69.9% vs. 33.1% for thrombocytopenia, P = 0.02). More patients with kidney from deceased donors received thymoglobulin in their immunosuppressive regimen (82% vs. 37%; P < 0.001). The median time to leukopenia and thrombocytopenia were 3 days and 1 day, respectively. Conclusion: Among immunosuppressive and prophylactic antimicrobial agents, thymoglobulin is more related to cytopenia; therefore, thymoglobulin dose reduction is recommended as the first intervention to manage cytopenia without need for reduction of its cumulative dose. The higher prevalence of cytopenia among recipients from deceased donors may be related to the higher use of thymoglobulin in these patients

Secular trends in the national prevalence of overweight and obesity during 2007-2009 in 6-year-old Iranian children

Background: This study aimed to determine the secular trends in the national prevalence of overweight and obesity among 6-year-old Iranian children, and to compare the results in Northern, Central and Southern parts of the country. Methods: The data were collected as part of a routine and mandatory national screening program on children entering elementary schools in 2007, 2008 and 2009. Results: The study population comprised 2,600,065 children including 862,433 in 2007, 782,244 in 2008 and 955,388 in 2009. Of total children 12.8%, 13.5% and 10.9% were overweight in 2007, 2008 and 2009, respectively (P > 0.05). The corresponding figures for obesity were 3.4%, 3.5% and 3.4%, respectively (P > 0.05). In all surveys, the prevalence of overweight was higher in Southern region than in the other two regions. P for trend was not significant for prevalence rates of overweight and obesity in any of the regions. Conclusions: To the best of our knowledge, this study was the first of its kind in presenting the nationwide trend of overweight and obesity in young children living in a developing country. It showed a considerably high prevalence of overweight and obesity, but with a constant rate in three years. The higher prevalence of overweight in Southern region than in Central and Northern regions might be related to the lower socioeconomic position of this population. At a very young age, children′s lifestyle is more under control of parents. Primordial and primary prevention efforts against the overweight epidemic can be effective and shall be further strengthened

Epigenetic regulators controlling osteogenic lineage commitment and bone formation

Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineage-specific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). This narrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for self-renewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesis.</p

G-protein coupled receptor 5C (GPRC5C) is required for osteoblast differentiation and responds to EZH2 inhibition and multiple osteogenic signals

Osteoblast differentiation is epigenetically suppressed by the H3K27 methyltransferase EZH2, and induced by the morphogen BMP2 and transcription factor RUNX2. These factors also regulate distinct G protein coupled receptors (GPRCs; e.g., PTH1R, GPR30/GPER1). Because GPRCs transduce many physiological stimuli, we examined whether BMP2 or EZH2 inhibition (i.e., GSK126) regulates other GPRC genes in osteoblasts. RNA-seq screening of &gt;400 mouse GPRC-related genes showed that many GPRCs are downregulated during osteogenic differentiation. The orphan receptor GPRC5C, along with a small subset of other GPRCs, is induced by BMP2 or GSK126 during Vitamin C dependent osteoblast differentiation, but not by all-trans retinoic acid. ChIP-seq analysis revealed that GSK126 reduces H3K27me3 levels at the GPRC5C gene locus in differentiating MC3T3-E1 osteoblasts, consistent with enhanced GPRC5C mRNA expression. Loss of function analyses revealed that shRNA-mediated depletion of GPRC5C decreases expression of bone markers (e.g., BGLAP and IBSP) and mineral deposition in response to BMP2 or GSK126. GPRC5C mRNA was found to be reduced in the osteopenic bones of KLF10 null mice which have compromised BMP2 signaling. GPRC5C mRNA is induced by the bone-anabolic activity of 17β-estradiol in trabecular but not cortical bone following ovariectomy. Collectively, these findings suggest that GPRC5C protein is a key node in a pro-osteogenic axis that is normally suppressed by EZH2-mediated H3K27me3 marks and induced during osteoblast differentiation by GSK126, BMP2, and/or 17β-estradiol. Because GPRC5C protein is an understudied orphan receptor required for osteoblast differentiation, identification of ligands that induce GPRC5C signaling may support therapeutic strategies to mitigate bone-related disorders.</p

The lysine methyltransferases SET and MYND domain containing 2 (Smyd2) and Enhancer of Zeste 2 (Ezh2) co-regulate osteoblast proliferation and mineralization

Bone formation is controlled by histone modifying enzymes that regulate post-translational modifications on nucleosomal histone proteins and control accessibility of transcription factors to gene promoters required for osteogenesis. Enhancer of Zeste homolog 2 (EZH2/Ezh2), a histone H3 lysine 27 (H3K27) methyl transferase, is a suppressor of osteoblast differentiation. Ezh2 is regulated by SET and MYND domain-containing protein 2 (SMYD2/Smyd2), a lysine methyltransferase that modifies both histone and non-histone proteins. Here, we examined whether Smyd2 modulates Ezh2 suppression of osteoblast differentiation. Musculoskeletal RNA-seq data show that SMYD2/Smyd2 is the most highly expressed SMYD/Smyd member in human bone tissues and mouse osteoblasts. Smyd2 loss of function analysis in mouse MC3T3 osteoblasts using siRNA depletion enhances proliferation and calcium deposition. Loss of Smyd2 protein does not affect alkaline phosphatase activity nor does it result in a unified expression response for standard osteoblast-related mRNA markers (e.g., Bglap, Ibsp, Spp1, Sp7), indicating that Smyd2 does not directly control osteoblast differentiation. Smyd2 protein depletion enhances levels of the osteo-suppressive Ezh2 protein and H3K27 trimethylation (H3K27me3), as expected from increased cell proliferation, while elevating the osteo-inductive Runx2 protein. Combined siRNA depletion of both Smyd2 and Ezh2 protein is more effective in promoting calcium deposition when compared to loss of either protein. Collectively, our results indicate that Smyd2 inhibits proliferation and indirectly the subsequent mineral deposition by osteoblasts. Mechanistically, Smyd2 represents a functional epigenetic regulator that operates in parallel to the suppressive effects of Ezh2 and H3K27 trimethylation on osteoblast differentiation