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.

Cellular Senescence in Human Skin Aging: Leveraging Senotherapeutics.

BACKGROUND: As the largest organ in the human body, the skin is continuously exposed to intrinsic and extrinsic stimuli that impact its functionality and morphology with aging. Skin aging entails dysregulation of skin cells and loss, fragmentation, or fragility of extracellular matrix fibers that are manifested macroscopically by wrinkling, laxity, and pigmentary abnormalities. Age-related skin changes are the focus of many surgical and nonsurgical treatments aimed at improving overall skin appearance and health. SUMMARY: As a hallmark of aging, cellular senescence, an essentially irreversible cell cycle arrest with apoptosis resistance and a secretory phenotype, manifests across skin layers by affecting epidermal and dermal cells. Knowledge of skin-specific senescent cells, such as melanocytes (epidermal aging) and fibroblasts (dermal aging), will promote our understanding of age-related skin changes and how to optimize patient outcomes in esthetic procedures. KEY MESSAGES: This review provides an overview of skin aging in the context of cellular senescence and discusses senolytic intervention strategies to selectively target skin senescent cells that contribute to premature skin aging.

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.

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 >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 17ß-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 17ß-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.

A chronic wound model to investigate skin cellular senescence.

Wound healing is an essential physiological process for restoring normal skin structure and function post-injury. The role of cellular senescence, an essentially irreversible cell cycle state in response to damaging stimuli, has emerged as a critical mechanism in wound remodeling. Transiently-induced senescence during tissue remodeling has been shown to be beneficial in the acute wound healing phase. In contrast, persistent senescence, as observed in chronic wounds, contributes to delayed closure. Herein we describe a chronic wound murine model and its cellular senescence profile, including the senescence-associated secretory phenotype.

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.

Clinical and Genetic Study of X-linked Agammaglobulinemia Patients (The Benefit of Early Diagnosis).

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by genetic defects in the Bruton tyrosine kinase (Btk) gene. XLA is characterized as an antibody deficiency by recurrent bacterial infections, the absence of peripheral B cells, and profound reductions in all immunoglobulin isotypes. This study aims to report the clinical and genetic features of five Iranian patients with XLA. Five male cases with recurrent bacterial infection entered this study based on clinical evaluation and Immunological screening tests. The levels of T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) were also measured in dried blood spot (DBS) samples. Sanger sequencing was applied to PCR products of DNA samples of the patients for genetic studies. All patients were from unrelated families with a mean age of 6.7 years (2.5-11) at the time of diagnosis with 4.8 mean years of delay in diagnosis. The most frequent clinical manifestations were recurrent respiratory infections and arthritis. In these patients, five previously reported mutations were found including four mutations (p.Q496X, p.Q497X, p.R520X, and p.R641H) in the Kinase domain besides one mutation (p.L37P) in the pleckstrin homology (PH) domain. Evaluations of KREC and TREC level in patients' DBS showed low-to-undetectable copies of KREC (0-2 copies/3.2mm DBS) with normal copies of TREC. As patients with XLA have complete immunoglobulin defects and develop severe and recurrent infections, early diagnosis would be beneficial for the improvement of their quality of life. The study results may provide valuable information for the diagnosis, genetic counseling and prenatal diagnosis for the patients and their family members and emphasize performing KREC as an early diagnostic test in patients with XLA.

Novel LAMA2 Gene Mutations Associated with Merosin-Deficient Congenital Muscular Dystrophy

Background: Merosin-deficient congenital muscular dystrophy (MDC1A) is a rare autosomal recessive genetic disease occurred due to mutations in the LAMA2 gene. This study investigated the molecular genetics of three Iranian MDC1A patients who manifested hypotonia, muscle weakness at birth, elevated levels of creatine kinase, and normal magnetic resonance imaging before the age of six months Methods: Peripheral blood samples were collected from three unrelated patients and their families after obtaining informed written consents. Genomic DNA was extracted and sequenced using next-generation sequencing, followed by Sanger confirmation. Results: Sequencing results revealed a known missense mutation, c.8665G>A, and two novel heterozygous sequencing variants affecting splicing, c.397-4_c.478del and c.7452-1G>A, in the LAMA2 gene. Reverse transcriptase-PCR analysis showed that a new intronic variant, c.7452-1G>A, produced aberrant splicing pattern in the patient. Conclusion: This study expands the mutation spectrum of LAMA2 and assists in the diagnosis, genetic counseling, and prenatal diagnosis of the affected families.