Differential display of human marrow stromal cells reveals unique mRNA expression patterns in response to dexamethasone

Abstract

Human bone marrow stromal cells (hBMSC) are pluripotent cells that have the ability to differentiate into bone, cartilage, hematopoietic-supportive stroma, and adipocytes in a process modulated by dexamethasone (DEX). To characterize changes in hBMSC in response to DEX, we carried out differential display experiments using hBMSC cultured for 1 week in the presence or absence of 10(-8) M DEX. When RNA from these cells was used for differential display, numerous cDNA bands were identified that were up-regulated and down-regulated by DEX. The cDNA bands were reamplified by PCR and directly used to screen an hBMSC cDNA library. Seven clones were isolated and characterized by DNA sequencing and found to encode the following genes: transforming growth factor-beta-induced gene product ((beta)ig-h3), calphobindin II, cytosolic thyroid-binding protein, 22-kDA smooth muscle protein (SM22), and the extracellular matrix proteins osteonectin/SPARC, type III collagen, and fibronectin. To confirm that these genes were regulated by DEX, the cells were treated continuously with this hormone for periods ranging from 2 to 30 days, and steady-state mRNA levels were measured by Northern blot analysis. All genes showed some level of regulation by DEX. The most profound regulation by DEX was observed in the (beta)ig-h3 gene, which showed a relative 10-fold decrease in mRNA levels after 6 days of treatment. Interestingly, (beta)ig-h3 expression was not altered by DEX in fibroblasts from other human tissues, including thymus stromal fibroblasts, spleen stromal fibroblasts, and foreskin fibroblasts. In summary, differential display of DEX-treated hBMSC revealed unique patterns of gene expression and has provided new information about phenotypic changes that accompany the differentiation of hBMSC toward osteogenesis. J. Cell. Biochem. 76:231-243, 1999. Published 1999 Wiley-Liss, Inc.

A transforming growth factor–β–induced protein stimulates endocytosis and is up-regulated in immature dendritic cells

“…40 Although our results suggest a role for ␤ig-h3 in the regulation of macrophage endocytosis, previous studies suggest a role of ␤ig-h3 as an ECM protein that regulates cell adhesion, differentiation, and proliferation. 27,[41][42][43][44] Our demonstration of the lack of disulfide-linked ␤ig-h3 oligomerization is also in contrast with previous detection of native tissue ␤ig-h3 as highly oligomerized fibrils. It is possible that ␤ig-h3 is found in ECM-associated and soluble forms, depending on the tissue compartments or cell types where it is produced, and the 2 forms may exhibit distinct functional properties.…”

RGD peptides released from βig-h3, a TGF-β-induced cell-adhesive molecule, mediate apoptosis

“…Introduction big-h3 is an extracellular matrix protein whose expression in several cell types, including human melanoma cells, mammary epithelial cells, keratinocytes, lung fibroblasts, and bone marrow stromal cells, can be induced by transforming growth factor-b (TGF-b) (Skonier et al, 1994;Dieudonne et al, 1999). The protein contains an RGD motif and four internal repeat domains that have highly conserved sequences also found in various secretory and membrane proteins from several species, including mammals, insects, sea urchins, plants, yeast, and bacteria (Kawamoto et al, 1998).…”

“…The protein contains an RGD motif and four internal repeat domains that have highly conserved sequences also found in various secretory and membrane proteins from several species, including mammals, insects, sea urchins, plants, yeast, and bacteria (Kawamoto et al, 1998). A number of studies have suggested that big-h3 is involved in cell growth (Skonier et al, 1994), cell differentiation (Dieudonne et al, 1999;Kim et al, 2000a), wound healing (Rawe et al, 1997), and cell adhesion (LeBaron et al, 1995;Ohno et al, 1999). Although the underlying mechanisms driving these effects have not been defined, we recently reported that big-h3 may mediate cell adhesion by interacting with the a3b1 integrin through two motifs residing within the 2nd and 4th repeat domains (Kim et al, 2000b).…”

Exequibilidade do DDRT-PCR para análise da expressão gênica diferencial em células de medula óssea murina

“…On the other hand, the two bands observed for the cultivated cells may be related with the process of cellular differentiation in vitro, and enrichment of mesenchymal cells due to culture conditions (DEMEN 10% SBF). 21,22,23 However, other studies suggest that the loss of constitutive expression for some genes by in vitro culture methods, which would be normally expressed in vivo, may be occurring. 20 In this work, the authors showed in their results that the gene for myeloperoxidase (bactericidal enzyme found primarily in neutrophils and monocytes) is expressed in bone in vivo, but its expression was not detected in in vitro culture of osteoblasts.…”