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Biomedical subjects

Dervis A M Salih

Publications and source records attributed to Dervis A M Salih.

5 recordsLinked to original sources

Convergence of Igf2 expression and adhesion signalling via RhoA and p38 MAPK enhances myogenic differentiation.

Cell-cell contact is essential for appropriate co-ordination of development and it initiates significant signalling events. During myogenesis, committed myoblasts migrate to sites of muscle formation, align and form adhesive contacts that instigate cell-cycle exit and terminal differentiation into multinucleated myotubes; thus myogenesis is an excellent paradigm for the investigation of signals derived from cell-cell contact. PI3-K and p38 MAPK are both essential for successful myogenesis. Pro-myogenic growth factors such as IGF-II activate PI3-K via receptor tyrosine kinases but the extracellular cues and upstream intermediates required for activation of the p38 MAPK pathway in myoblast differentiation are not known. Initial observations suggested a correlation between p38 MAPK phosphorylation and cell density, which was also related to N-cadherin levels and Igf2 expression. Subsequent studies using N-cadherin ligand, dominant-negative N-cadherin, constitutively active and dominant-negative forms of RhoA, and MKK6 and p38 constructs, reveal a novel pathway in differentiating myoblasts that links cell-cell adhesion via N-cadherin to Igf2 expression (assessed using northern and promoter-reporter analyses) via RhoA and p38alpha and/or beta but not gamma. We thus define a regulatory mechanism for p38 activation that relates cell-cell-derived adhesion signalling to the synthesis of the major fetal growth factor, IGF-II.

Animals↗

Insulin-like growth factor-binding protein-5 induces a gender-related decrease in bone mineral density in transgenic mice.

IGF-binding protein-5 (IGFBP-5) is abundant in serum and bone during normal skeletal development, but levels decrease in osteoporosis. Studies have shown that IGFBP-5 stimulates markers of bone formation by potentiating IGF actions and by IGF-independent actions. To test the hypothesis that IGFBP-5 promotes the acquisition of bone mineral density (BMD), we generated transgenic (Tg) mice overexpressing Igfbp5 using a cytomegalovirus enhancer and beta-actin promoter (CMV/betaA). Tg animals showed an increase in serum IGFBP-5 concentrations by 7.7- to 3.5-fold at 3-8 wk of age, respectively. Concentrations were 6-49% higher for males compared with females in both wild-type and Tg mice. Surprisingly, BMD decreased in a gender-dependent manner, with Tg male adults affected more severely than Tg females (31.3% vs. 19.2% reduction, respectively, compared with wild-type mice, assessed by dual energy x-ray absorptiometry). Significant gender differences in BMD were confirmed by peripheral quantitative computed tomography. Histomorphometry revealed that although the bone formation rate and mineralizing surface at the periosteum decreased in Tg mice, they increased at the endosteum, suggesting opposing effects of IGFBP-5 on periosteal and endosteal osteoblasts (by altering proliferation or survival). These findings differ from previous observations in Igf1- and Igf2-null animals. In conclusion, IGFBP-5 has a significant influence on BMD acquisition and maintenance that is dependent on gender and age. The phenotype of Igfbp5 mice cannot be explained solely by IGF inhibition; thus, this study provides the first in vivo evidence, by genetic manipulation, for IGF-independent actions of IGFBP-5 in bone function. These findings have implications for the gender-biased progression of osteoporosis.

Absorptiometry, Photon↗

Insulin-like growth factor-binding protein 5 (Igfbp5) compromises survival, growth, muscle development, and fertility in mice.

The insulin-like growth factors (IGFs) are essential for development; bioavailable IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). Igfbp5 is the most conserved and is developmentally up-regulated in key lineages and pathologies; in vitro studies suggest that IGFBP-5 functions independently of IGF interaction. Genetic ablation of individual Igfbps has yielded limited phenotypes because of substantial compensation by remaining family members. Therefore, to reveal Igfbp5 actions in vivo, we generated lines of transgenic mice that ubiquitously overexpressed Igfbp5 from early development. Significantly increased neonatal mortality, reduced female fertility, whole-body growth inhibition, and retarded muscle development were observed in Igfbp5-overexpressing mice. The magnitude of the response in individual transgenic lines was positively correlated with Igfbp5 expression. Circulating IGFBP-5 concentrations increased a maximum of only 4-fold, total and free IGF-I concentrations increased up to 2-fold, and IGFBP-5 was detected in high M(r) complexes; however, no detectable decrease in the proportion of free IGF-I was observed. Thus, despite only modest changes in IGF and IGFBP concentrations, the Igfbp5-overexpressing mice displayed a phenotype more extreme than that observed for other Igfbp genetic models. Although growth retardation was obvious prenatally, maximal inhibition occurred postnatally before the onset of growth hormone-dependent growth, regardless of Igfbp5 expression level, revealing a period of sensitivity to IGFBP-5 during this important stage of tissue programming.

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Partitioning of IGFBP-5 actions in myogenesis: IGF-independent anti-apoptotic function.

Igfbp5 is upregulated during the differentiation of several key cell lineages and in some tumours; the function of IGFBP-5 in these physiological and pathological situations is unknown. Since IGFBP-5 contains sequence motifs consistent with IGF-independent actions, the aim of these studies was to distinguish between IGF-dependent and -independent actions of IGFBP-5. Myc-tagged wild-type (termed wtIGFBP-5) and non-IGF binding mouse Igfbp5 (termed mutIGFBP-5) cDNAs were generated and used to transfect C2 myoblasts, a cell line that undergoes differentiation to myotubes in an IGF- and IGFBP-5-regulated manner. WtIGFBP-5, but not mutIGFBP-5, inhibited myogenesis, as assessed by cell morphology, MHC immunocytochemistry and caveolin 3 expression. However, both wt- and mutIGFBP-5 increased cell survival and decreased apoptosis, as indicated by decreased caspase-3 activity and cell surface annexin V binding. Further examination of apoptotic pathways revealed that wt- and mutIGFBP-5 ameliorated the increase in caspase-9 but not the modest increase in caspase-8 during myogenesis, suggesting that IGFBP-5 increased cell survival via inhibition of intrinsic cell death pathways in an IGF-independent manner. The relationship between IGF-II and IGFBP-5 was examined further by cotransfecting C2 myoblasts with antisense Igf2 (previously established to induce increased cell death) and Igfbp5; both wt- and mutIGFBP-5 conferred equivalent protection against the decreased cell survival and increased apoptosis. In conclusion, we have partitioned IGFBP-5 action in myogenesis into IGF-dependent inhibition of differentiation and IGF-independent cell survival. Our findings suggest that, by regulation of cell survival, IGFBP-5 has an autonomous role in the regulation of cell fate in development and in tumourigenesis.

Animals↗

Akt2, a novel functional link between p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in myogenesis.

Activation of either the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt or the p38 mitogen-activated protein kinase (MAPK) signaling pathways accelerates myogenesis but only when the reciprocal pathway is functional. We therefore examined the hypothesis that cross-activation between these signaling cascades occurs to orchestrate myogenesis. We reveal a novel and reciprocal cross-talk and activation between the PI 3-kinase/Akt and p38 MAPK pathways that is essential for efficient myoblast differentiation. During myoblast differentiation, Akt kinase activity correlated with S473 but not T308 phosphorylation and occurred 24 h after p38 activation. Inhibition or activation of p38 with SB203580, dominant-negative p38, or MKK6EE regulated Akt kinase activity. Analysis of Akt isoforms revealed a specific increase in Akt2 protein levels that coincided with AktS473 phosphorylation during myogenesis and an enrichment of S473-phosphorylated Akt2. Akt2 promoter activity and protein levels were regulated by p38 activation, thus providing a mechanism for communication. Subsequent Akt activation by S473 phosphorylation was PI 3-kinase dependent and specific for Akt2 rather than Akt1. Complementary to p38-mediated transactivation of Akt, activation or inhibition of PI 3-kinase regulated p38 activity upstream of MKK6, demonstrating reciprocal communication and positive feedback characteristic of myogenic regulation. Our findings have identified novel communication between p38 MAPK and PI 3-kinase/Akt via Akt2.

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