Shift of syndecan-1 expression from epithelial to stromal cells during progression of solid tumours.

Syndecan-1 (SDC-1), a protein found on cells and in the extracellular matrix, participates in cell proliferation, cell migration and cell-matrix interactions. SDC-1 expression correlates with the maintenance of epithelial morphology and inhibition of invasiveness. In the present study, a second SDC-1 mRNA isoform was identified and the expression of both transcripts was investigated in various normal and malignant tissues. Both transcripts were coexpressed at equal levels in all tissues and organs analysed. Cancer-profiling array (CPA) analysis of 241 non-enriched tumour and normal cDNAs revealed stronger upregulation of SDC-1 in tumour tissues as compared with oligonucleotide array-based expression analysis of SDC-1 in microdissected breast, prostate, lung, and colon carcinoma cells. With in situ hybridisation and immunohistochemistry it was demonstrated that this difference in SDC-1 expression originates from stromal cells present in tumour connective tissue. But only the cells in connective tissue surrounding breast, lung, colon and bladder carcinoma showed upregulation of SDC-1. These stromal cells were characterised as spindle cells with myofibroblastic differentiation and they may contribute to the dedifferentiation of tumour cells and the development of metastasis.

Activation of myogenesis by the homeobox gene Lbx1 requires cell proliferation.

Myogenic differentiation can be initiated by a limited number of molecules. In this work, we analyzed the function of the homeobox gene Lbx1 in chicken embryos and explant cultures. We demonstrate that overexpression of Lbx1 in vivo and in vitro leads to a strong activation of various muscle markers. We show that cell proliferation, which is strongly stimulated by Lbx1 and Pax3, is required for Lbx1- or Pax3-dependent myogenic activation. Inhibition of cell proliferation prevents expression of muscle differentiation markers, while the activation of other putative downstream targets of Pax3 and Lbx1 is not affected. Our findings imply that a critical function of Pax3 and Lbx1 during muscle cell formation is the enlargement of muscle cell populations. The growth of the muscle precursor cell population may increase the bias for myogenic differentiation and thus enable myogenic cells to respond to environmental cues.

Sonic hedgehog is a survival factor for hypaxial muscles during mouse development.

Sonic hedgehog (Shh) has been proposed to function as an inductive and trophic signal that controls development of epaxial musculature in vertebrate embryos. In contrast, development of hypaxial muscles was assumed to occur independently of Shh. We here show that formation of limb muscles was severely affected in two different mouse strains with inactivating mutations of the Shh gene. The limb muscle defect became apparent relatively late and initial stages of hypaxial muscle development were unaffected or only slightly delayed. Micromass cultures and cultures of tissue fragments derived from limbs under different conditions with or without the overlaying ectoderm indicated that Shh is required for the maintenance of the expression of myogenic regulatory factors (MRFs) and, consecutively, for the formation of differentiated limb muscle myotubes. We propose that Shh acts as a survival and proliferation factor for myogenic precursor cells during hypaxial muscle development. Detection of a reduced but significant level of Myf5 expression in the epaxial compartment of somites of Shh homozygous mutant embryos at E9.5 indicated that Shh might be dispensable for the initiation of myogenesis both in hypaxial and epaxial muscles. Our data suggest that Shh acts similarly in both somitic compartments as a survival and proliferation factor and not as a primary inducer of myogenesis.

Heterozygous myogenic factor 6 mutation associated with myopathy and severe course of Becker muscular dystrophy.

Myogenic factors (MYF) belong to the basic helix-loop-helix (bHLH) transcription factor family and regulate myogenesis and muscle regeneration. The physiological importance of both functions was demonstrated in homozygous Myf knockout mice and mdx mice. Myf5 and Myod are predominantly expressed in proliferating myoblasts while Myf4 and Myf6 are involved in differentiation of myotubes. In a boy with myopathy and an increase of muscle fibres with central nuclei we detected a heterozygous 387G-->T nucleotide transversion in the MYF6 gene (MIM*159991). Protein-protein interaction of mutant MYF6 was reduced, and DNA-binding potential and transactivation capacity were abolished, thus demonstrating MYF6 haploinsufficiency. The boy's father carried the identical mutation and, in addition, an in-frame deletion of exons 45-47 in his dystrophin gene. This mutation is normally associated with a mild to moderate course of Becker muscular dystrophy but the father suffered from a severe course of Becker muscular dystrophy suggesting MYF6 as a modifier.

Two highly related homeodomain proteins, Nkx5-1 and Nkx5-2, display different DNA binding specificities.

The mouse Nkx5-1 and Nkx5-2 genes are related to NK genes in Drosophila and encode proteins with very similar homeodomains. In higher vertebrates Nkx5 genes are specifically expressed in the inner ear. Inactivation of the mouse Nkx5-1 gene by homologous recombination revealed a critical role for the formation of vestibular inner ear structures. Here, we investigated biochemical properties of the proteins encoded by the Nkx5 genes. A similar consensus binding sequence was isolated for both Nkx5 proteins using binding site selection. This sequence is related to target sequences previously identified for other Nkx proteins and contains the conserved homeodomain binding core TAAT. An additional, novel and unrelated high affinity binding sequence could be identified for the Nkx5-2 protein.

Pax-3 is necessary but not sufficient for lbx1 expression in myogenic precursor cells of the limb.

In vertebrates all skeletal muscles of trunk and limbs are derived from condensations of the paraxial mesoderm, the somites. Limb muscle precursor cells migrate during embryogenesis from somites to limb buds where migration stops and differentiation occurs. We have characterized lbx1 homeobox genes in chicken and mice and found them to be expressed in migrating limb muscle precursor cells in both species. Analysis of splotch mutant mice showed that lbx1 and c-met are differently affected by the lack of Pax-3. Limb buds of splotch (Pax-3 mutant) mice were devoid of lbx1 transcripts, while expression of c-met was still detectable at a low level. The presence of c-met-positive cells in splotch mice entering the limbs indicates that migration of cells from somites to limbs is not entirely dependent on Pax-3. We show that induction of epithelial to mesenchymal transition of Pax-3-positive cells by SF/HGF was not sufficient to induce ectopic lbx-1 expression at the inter-limb level, while ectopic limb formation was able to activate lbx1 expression. We postulate that Pax-3 is necessary for lbx1 expression in the lateral tips of somites but additional, yet unknown signals derived from limb buds are needed to initiate lbx1 expression. The role of limb bud-derived signals involved in targeted muscle precursor cell migration, and lbx1 activation was further confirmed by analysis of explanted somite/limb bud co-cultures in collagen gels.