Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy

Adult skeletal muscles adapt their fiber size to workload. We show that serum response factor (Srf) is required for satellite cell-mediated hypertrophic muscle growth. Deletion of Srf from myofibers and not satellite cells blunts overload-induced hypertrophy, and impairs satellite cell proliferation and recruitment to pre-existing fibers. We reveal a gene network in which Srf within myofibers modulates interleukin-6 and cyclooxygenase-2/interleukin-4 expressions and therefore exerts a paracrine control of satellite cell functions. In Srf-deleted muscles, in vivo overexpression of interleukin-6 is sufficient to restore satellite cell proliferation but not satellite cell fusion and overall growth. In contrast cyclooxygenase-2/interleukin-4 overexpression rescue satellite cell recruitment and muscle growth without affecting satellite cell proliferation, identifying altered fusion as the limiting cellular event. These findings unravel a role for Srf in the translation of mechanical cues applied to myofibers into paracrine signals, which in turn will modulate satellite cell functions and support muscle growth.     

E2F-1 is essential for normal epidermal wound repair.

E2F factors are involved in proliferation and apoptosis. To understand the role of E2F-1 in the epidermis, we screened wild type and E2F-1(-/-) keratinocyte mRNA for genes differentially expressed in the two cell populations. We demonstrate the reduced expression of integrins alpha(5), alpha(6), beta(1), and beta(4) in E2F-1(-/-) keratinocytes associated with reduced activation of Jun terminal kinase and Erk upon integrin stimulation. As a consequence of altered integrin expression and function, E2F-1(-/-) keratinocytes also show impaired migration, adhesion to extracellular matrix proteins, and a blunted chemotactic response to transforming growth factor-gamma1. E2F-1(-/-) keratinocytes, but not dermal fibroblasts, exhibit altered patterns of proliferation, including significant delays in transit through both G(1) and S phases of the cell cycle. Recognizing that proliferation and migration are key for proper wound healing in vivo, we postulated that E2F-1(-/-) mice may exhibit abnormal epidermal repair upon injury. Consistent with our hypothesis, E2F-1(-/-) mice exhibited impaired cutaneous wound healing. This defect is associated with substantially reduced local inflammatory responses and rates of re-epithelialization. Thus, we demonstrate that E2F-1 is indispensable for a hitherto unidentified cell type-specific and unique role in keratinocyte proliferation, adhesion, and migration as well as in proper wound repair and epidermal regeneration in vivo.     

Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange

Elk-1 is a member of the TCF subfamily of Ets proteins. TCFs interact with SRF at serum response elements (SREs) of immediate early genes (IEGs), such as c-fos and Egr-1, thereby mediating IEG induction upon extracellular stimulation. We previously generated an Elk-1 null allele (Elk1-137) in murine embryonic stem (ES) cells by homologous recombination. In Elk1-137, the Elk-1 gene was replaced by a Hygromycin B phosphotransferase - Thymidine Kinase (HygTk) fusion gene, flanked by two nonidentical Flp recombinase recognition (FRT) sites (Cesari et al., [2004] Mol Cell Biol, in press) to allow for the subsequent generation of alternative alleles of interest by recombinase-mediated cassette exchange (RMCE). Elk1-deficient mice derived from Elk-1((137/0)) ES cells are viable and do not reveal strong phenotypical abnormalities, apart from male sterility. However, the Elk-1 locus contains the Tk cassette, which has previously been related to this defect. Therefore, in our first experiment involving the technique of Flp RMCE we chose to remove the HygTk cassette in Elk-1((137/0)) ES cells and to generate Elk-1((RMCE16/0)) and Elk-1((RMCE16/RMCE16)) mice. In so doing, we provide evidence that the sterility of Elk1((137/0)) mice was not due to the absence of Elk-1 but rather the presence of HygTk. This is the first report of mice derived from ES cells which were subjected to Flp RMCE and thus proves that RMCE is a powerful tool for the genetic engineering of previously tagged loci in the mouse genome.     

Adenosine stimulation of proliferation of breast carcinoma cell lines: evaluation of the [3H]thymidine assay system and modulatory effects of the cellular microenvironment in vitro

The purine nucleoside adenosine is produced at increased levels in the tissues of solid cancers as a result of local hypoxia. Adenosine inhibits the cell-mediated anti-tumor immune response, promotes tumor cell migration and angiogenesis, and stimulates the proliferation of tumor cells. We examined the stimulatory effect of adenosine on DNA synthesis, cell cycle progression, and cell proliferation in MCF7 and T-47D breast carcinoma cell lines in culture, and identified factors that modulate the growth response. The ability of adenosine to stimulate DNA synthesis, as measured by the incorporation of [(3)H]thymidine, was independent of the total radioactivity of the [(3)H]thymidine up to 10 microCi/ml, total thymidine concentrations up to 100 microM, and the labeling interval. It was also not affected by the presence of low-molecular-weight compounds (such as thymidine and adenosine) in the serum used to supplement the medium. Adenosine stimulated DNA synthesis and cell proliferation with an EC(50) of 4-6 microM and a maximum response at 30-100 microM, when given as a single addition. The stimulatory effect of adenosine involved progression through the cell cycle and a genuine increase in cell number, in the absence of significant apoptotic or necrotic cell death. The mitogenic effect of adenosine was dependent upon the culture cell density, with an optimum adenosine response at around 50% of confluent density. The response was also highly dependent upon the form of the serum addition to the growth medium, with the best response elicited in the presence of low concentrations of nonfetal bovine serum, although adenosine was mitogenic under standard culture conditions. The effects of serum supplementation and cell density were not due to differences in the rate of adenosine metabolism by either serum or cellular enzymes, but appeared to result from changes in the sensitivity to adenosine of the cell population in response to environmental cues. We, therefore, find that adenosine is consistently mitogenic for human breast carcinoma cells, and that the [(3)H]thymidine incorporation assay is a valid measure of this response. The data are consistent with the stimulatory effect of adenosine on cell proliferation being modulated by the local cellular environment.     

Egr-1 and serum response factor are involved in growth factors- and serum-mediated induction of E2-EPF UCP expression that regulates the VHL-HIF pathway

E2-EPF ubiquitin carrier protein (UCP) has been shown to be highly expressed in common human cancers and target von Hippel-Lindau (VHL) for proteosomal degradation in cells, thereby stabilizing hypoxia-inducible factor (HIF)-1alpha. Here, we investigated cellular factors that regulate the expression of UCP gene. Promoter deletion assay identified binding sites for early growth response-1 (Egr-1) and serum response factor (SRF) in the UCP promoter. Hepatocyte or epidermal growth factor (EGF), or phorbol 12-myristate 13-acetate induced UCP expression following early induction of Egr-1 expression in HeLa cells. Serum increased mRNA and protein levels of SRF and UCP in the cell. By electrophoretic mobility shift and chromatin immunoprecipitation assays, sequence-specific DNA-binding of Egr-1 and SRF to the UCP promoter was detected in nuclear extracts from HeLa cells treated with EGF and serum, respectively. Overexpression of Egr-1 or SRF increased UCP expression. RNA interference-mediated depletion of endogenous Egr-1 or SRF impaired EGF- or serum-mediated induction of UCP expression, which was required for cancer cell proliferation. Systemic delivery of EGF into mice also increased UCP expression following early induction of Egr-1 expression in mouse liver. The induced UCP expression by the growth factors or serum increased HIF-1alpha protein level under non-hypoxic conditions, suggesting that the Egr-1/SRF-UCP-VHL pathway is in part responsible for the increased HIF-1alpha protein level in vitro and in vivo. Thus, growth factors and serum induce expression of Egr-1 and SRF, respectively, which in turn induces UCP expression that positively regulates cancer cell growth.     

Serum response factor controls CYLD expression via MAPK signaling pathway

Tumor suppressor gene CYLD is a deubiquitinating enzyme which negatively regulates various signaling pathways by removing the lysine 63-linked polyubiquitin chains from several specific substrates. Loss of CYLD in different types of tumors leads to either cell survival or proliferation. In this study we demonstrate that lack of CYLD expression in CYLD-/- MEFs increases proliferation rate of these cells compared to CYLD+/+ in a serum concentration dependent manner without affecting cell survival. The reduced proliferation rate in CYLD+/+ in the presence of serum was due to the binding of serum response factor (SRF) to the serum response element identified in the CYLD promoter for the up-regulation of CYLD levels. The serum regulated recruitment of SRF to the CYLD promoter was dependent on p38 mitogen-activated protein kinase (MAPK) activity. Elimination of SRF by siRNA or inhibition of p38 MAPK reduced the expression level of CYLD and increased cell proliferation. These results show that SRF acts as a positive regulator of CYLD expression, which in turn reduces the mitogenic activation of serum for aberrant proliferation of MEF cells.     

Effects of simulated microgravity on embryonic stem cells

There have been many studies on the biological effects of simulated microgravity (SMG) on differentiated cells or adult stem cells. However, there has been no systematic study on the effects of SMG on embryonic stem (ES) cells. In this study, we investigated various effects (including cell proliferation, cell cycle distribution, cell differentiation, cell adhesion, apoptosis, genomic integrity and DNA damage repair) of SMG on mouse embryonic stem (mES) cells. Mouse ES cells cultured under SMG condition had a significantly reduced total cell number compared with cells cultured under 1 g gravity (1G) condition. However, there was no significant difference in cell cycle distribution between SMG and 1G culture conditions, indicating that cell proliferation was not impaired significantly by SMG and was not a major factor contributing to the total cell number reduction. In contrast, a lower adhesion rate cultured under SMG condition contributed to the lower cell number in SMG. Our results also revealed that SMG alone could not induce DNA damage in mES cells while it could affect the repair of radiation-induced DNA lesions of mES cells. Taken together, mES cells were sensitive to SMG and the major alterations in cellular events were cell number expansion, adhesion rate decrease, increased apoptosis and delayed DNA repair progression, which are distinct from the responses of other types of cells to SMG.