Expression of proto-oncogenes and muscle specific genes during cardiac hypertrophy and development in rats and humans

A regulatory interdependence of expression of proto-oncogenes and muscle specific genes observed in smooth muscle was examined in cardiac muscle during normal development and hypertrophy both in rats and humans. During normal development in rats, myosin light chain 2 expression is very low at prenatal stages, while c-fos expression starts from the early stages of embryonic development. In aorta constricted rats c-fos induction occurs within 30 min whereas myosin light chain 2 expression is sufficiently high only after 3 or 4 days of post operative period. In the case of humans, the expression of myosin light chain 2 as well as c-fos occurs at high levels during embryonic development. Similar results were obtained with tissue samples obtained from patients with cardiac abnormalities. Induction of the c-fos gene in cultured myocytes by 12-O-tetradeeanoylphorbol 13-acetate has no influence on the expression of myosin light chain 2. These studies were extended with studies on c-myc and Β-myosin heavy chain gene expression which revealed a similar pattern of expression as that of c-fos and myosin light chain 2. These results have indicated that the expression of proto-oncogenes in cardiac muscle may be independently regulated from the expression of muscle specific genes

Role of Matrix Metalloproteinase 13 in Both Endochondral and Intramembranous Ossification during Skeletal Regeneration

Extracellular matrix (ECM) remodeling is important during bone development and repair. Because matrix metalloproteinase 13 (MMP13, collagenase-3) plays a role in long bone development, we have examined its role during adult skeletal repair. In this study we find that MMP13 is expressed by hypertrophic chondrocytes and osteoblasts in the fracture callus. We demonstrate that MMP13 is required for proper resorption of hypertrophic cartilage and for normal bone remodeling during non-stabilized fracture healing, which occurs via endochondral ossification. However, no difference in callus strength was detected in the absence of MMP13. Transplant of wild-type bone marrow, which reconstitutes cells only of the hematopoietic lineage, did not rescue the endochondral repair defect, indicating that impaired healing in Mmp13−/− mice is intrinsic to cartilage and bone. Mmp13−/− mice also exhibited altered bone remodeling during healing of stabilized fractures and cortical defects via intramembranous ossification. This indicates that the bone phenotype occurs independently from the cartilage phenotype. Taken together, our findings demonstrate that MMP13 is involved in normal remodeling of bone and cartilage during adult skeletal repair, and that MMP13 may act directly in the initial stages of ECM degradation in these tissues prior to invasion of blood vessels and osteoclasts

GSK-3β Controls Osteogenesis through Regulating Runx2 Activity

Despite accumulated knowledge of various signalings regulating bone formation, the molecular network has not been clarified sufficiently to lead to clinical application. Here we show that heterozygous glycogen synthase kinase-3β (GSK-3β)-deficient mice displayed an increased bone formation due to an enhanced transcriptional activity of Runx2 by suppressing the inhibitory phosphorylation at a specific site. The cleidocranial dysplasia in heterozygous Runx2-deficient mice was significantly rescued by the genetic insufficiency of GSK-3β or the oral administration of lithium chloride, a selective inhibitor of GSK-3β. These results establish GSK-3β as a key attenuator of Runx2 activity in bone formation and as a potential molecular target for clinical treatment of bone catabolic disorders like cleidocranial dysplasia

Runx Expression Is Mitogenic and Mutually Linked to Wnt Activity in Blastula-Stage Sea Urchin Embryos

The Runt homology domain (Runx) defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation.Depletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU) incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR) studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP) indicates that Runx target sites within 5′ sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastula stage. Furthermore, experiments using a green fluorescent protein (GFP) reporter transgene show that the blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545–558, 2005) is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK)-3.These results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development

Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: an in vitro study

Background: Although pulsed electromagnetic field (PEMF) stimulation may be clinically beneficial during fracture healing and for a wide range of bone disorders, there is still debate on its working mechanism. Mesenchymal stem cells are likely mediators facilitating the observed clinical effects of PEMF. Here, we performed in vitro experiments to investigate the effect of PEMF stimulation on human bone marrow-derived stromal cell (BMSC) metabolism and, specifically, whether PEMF can stimulate their osteogenic differentiation. Methods: BMSCs derived from four different donors were cultured in osteogenic medium, with the PEMF treated group being continuously exposed to a 15 Hz, 1 Gauss EM field, consisting of 5-millisecond bursts with 5-microsecond pulses. On culture day 1, 5, 9, and 14, cells were collected for biochemical analysis (DNA amount, alkaline phosphatase activity, calcium deposition), expression of various osteoblast-relevant genes and activation of extracellular signal-regulated kinase (ERK) signaling. Differences between treated and control groups were analyzed using the Wilcoxon signed rank test, and considered significant when p < 0.05. Results: Biochemical analysis revealed significant, differentiation stage-dependent, PEMF-induced differences: PEMF increased mineralization at day 9 and 14, without altering alkaline phosphatase activity. Cell proliferation, as measured by DNA amounts, was not affected by PEMF until day 14. Here, DNA content stagnated in PEMF treated group, resulting in less DNA compared to control. Quantitative RT-PCR revealed that during early culture, up to day 9, PEMF treatment increased mRNA levels of bone morphogenetic protein 2, transforming growth factor-beta 1, osteoprotegerin, matrix metalloproteinase-1 and-3, osteocalcin, and bone sialoprotein. In contrast, receptor activator of NF-B ligand expression was primarily stimulated on day 14. ERK1/2 phosphorylation was not affected by PEMF stimulation. Conclusions: PEMF exposure of differentiating human BMSCs enhanced mineralization and seemed to induce differentiation at the expense of proliferation. The osteogenic stimulus of PEMF was confirmed by the up-regulation of several osteogenic marker genes in the PEMF treated group, which preceded the deposition of mineral itself. These findings indicate that PEMF can directly stimulate osteoprogenitor cells towards osteogenic differentiation. This supports the theory that PEMF treatment may recruit these cells to facilitate an osteogenic response in vivo. © 2010 Jansen et al; licensee BioMed Central Ltd

MAPK ERK Signaling Regulates the TGF-β1-Dependent Mosquito Response to Plasmodium falciparum

Malaria is caused by infection with intraerythrocytic protozoa of the genus Plasmodium that are transmitted by Anopheles mosquitoes. Although a variety of anti-parasite effector genes have been identified in anopheline mosquitoes, little is known about the signaling pathways that regulate these responses during parasite development. Here we demonstrate that the MEK-ERK signaling pathway in Anopheles is controlled by ingested human TGF-β1 and finely tunes mosquito innate immunity to parasite infection. Specifically, MEK-ERK signaling was dose-dependently induced in response to TGF-β1 in immortalized cells in vitro and in the A. stephensi midgut epithelium in vivo. At the highest treatment dose of TGF-β1, inhibition of ERK phosphorylation increased TGF-β1-induced expression of the anti-parasite effector gene nitric oxide synthase (NOS), suggesting that increasing levels of ERK activation negatively feed back on induced NOS expression. At infection levels similar to those found in nature, inhibition of ERK activation reduced P. falciparum oocyst loads and infection prevalence in A. stephensi and enhanced TGF-β1-mediated control of P. falciparum development. Taken together, our data demonstrate that malaria parasite development in the mosquito is regulated by a conserved MAPK signaling pathway that mediates the effects of an ingested cytokine