Myocyte enhancer factor 2C: an osteoblast transcription factor identified by DMSO enhanced mineralization

Free to read on publisher website Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem-cell mediated therapies for fracture and other orthopaedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of simulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase (ALP) activity and extracellular matrix mineralization. Furthermore, similar DMSO mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1 we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased ALP activity and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. Flow on knockdown of bone specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts

Expression of the chemokine receptor CCR6 in human renal inflammation

Background. Nodular inflammatory cell infiltrates with defined microarchitecture, i.e. tertiary lymphoid organs, develop in the tubulointerstitium during chronic renal inflammation. CCR6 and the corresponding ligand CCL20 are involved in the formation of gut-associated lymphatic tissue. We hypothesized that CCR6 might be involved in the formation of nodular infiltrates in the kidney. Methods. CCR6- and CD20-positive B cells were localized in renal biopsies with IgA nephropathy (n = 13), membranous nephropathy (n = 12), crescentic glomerulonephritis (cGN, n = 11) and chronic interstitial nephritis (n = 13), and in pre-implantation biopsies as controls (n = 8). The mRNA expression of CCR6 and the ligand CCL20 was quantified by real-time RT-PCR in 51 renal biopsies of the same disease entities. Results. In the pre-transplant biopsies, CCR6 was expressed by endothelial cells of peritubular and glomerular capillaries. In patients with glomerulonephritis, infiltrating cells were positive particularly in areas of nodular inflammatory cell accumulations. A major part of the CCR6-positive cells were CD20-positive B cells, but a part of the CD3-positive T cells were also found to be positive. The constitutive expression of CCR6 on the endothelium of glomerular capillaries was lost in biopsies with progressive injury. Tubular epithelial cells expressed CCR6 in inflamed kidneys, most commonly on the basolateral side. Conclusions. CCR6 and the corresponding ligand CCL20 might therefore be involved in the recruitment of T and B cells to organized nodular infiltrates in chronic renal inflammation. The functional role of endothelial CCR6 needs to be evaluated in further studie

Proliferation of Acid-Secretory Cells in the Kidney during Adaptive Remodelling of the Collecting Duct

The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H+-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H+-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH4Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH4Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions

Remodelling and PCNA positive cell counts.

<p>(<b>A–C</b>) Summary of cells counts assessing the relative abundance of AE1, pendrin or AQP2 positive cells in the CNT, CCD, and OMCD of rats treated for 12 hrs, 4 or 7 days with NH₄Cl in the drinking water. (<b>D–F</b>) Percentage of AE1, pendrin, or AQP2 positive cells that were also stained for PCNA under the different conditions. * marks significant difference compared to control.</p

Early proliferation during induction and adaption to metabolic acidosis.

<p>Appearance of PCNA positive type A intercalated cells during acidosis. Kidney sections were stained with antibodies against either the type A intercalated cell specific AE1 protein (green), or the non-type A intercalated cell specific pendrin protein (red, asterisk), and antibodies against PCNA (red nuclei). (<b>A</b>) In the OMCD, PCNA labelling was seen in single cells not stained for AE1 under control conditions (arrow). (<b>B</b>) Co-staining of PCNA was seen in a few AE1 positive cells in the OMCD after 12 hrs NH₄Cl-loading (arrow).</p

Proliferation of segment-specific cells in the outer medullary collecting duct (OMCD).

<p>Kidney sections were stained with antibodies against the AQP2 water channel (green), a marker of segment-specific cells, and against BrdU (red). Colocalization of both stainings was observed in cells of animal groups in the OMCD (arrows).</p

Proliferation of segment specific cells in the connecting tubule (CNT).

<p>Kidney sections were stained with antibodies against calbindin D28k (green), a marker of segment-specific cells in the CNT, and against BrdU (red). Colocalization of both stainings was observed in cells of animal groups in the CNT (arrows).</p

Proliferation of type A intercalated cells during metabolic acidosis.

<p>Kidney sections were stained with antibodies against AE1 (green), pendrin (red, asterisk), and BrdU (red nuclei). (<b>A–E</b>) Localization of BrdU positive cells in the CCD of control rats (A) or animals on NaCl for 7 days (B), NH₄Cl for 4 or 7 days (C,D), or receiving acetazolamide for 10 days (E). BrdU colocalized with AE1 in NH₄Cl or acetazolamide treated rats (arrows). (<b>F–K</b>) Colocalization of BrdU and AE1 staining (arrows) in OMCD intercalated cells in rats treated with NH₄Cl (H, I) or acetazolamide (K) but not in control (F) or NaCl treated (G).</p

BrdU labeled cells.

<p>Data are given as mean ± SEM.</p><p>*marks significant difference between control and treated group.</p