Biomedical subjects

M Reza Seghatoleslami

Publications and source records attributed to M Reza Seghatoleslami.

6 recordsLinked to original sources

PubMed2005

Heparan sulfate proteoglycans including syndecan-3 modulate BMP activity during limb cartilage differentiation.

Bone morphogenetic proteins (BMPs) are involved in multiple aspects of limb development including regulation of cartilage differentiation. Several BMPs bind strongly to heparin, and heparan sulfate proteoglycans (HSPGs) at the cell surface or in the extracellular matrix have recently been implicated as modulators of BMP signaling in some developing systems. Here we have explored the role of HSPGs in regulating BMP activity during limb chondrogenesis by evaluating the effects of exogenous heparan sulfate (HS), heparitinase treatment, and overexpression of the HSPG syndecan-3 on the ability of BMP2 to modulate the chondrogenic differentiation of limb mesenchymal cells in micromass culture. Exogenous HS dramatically enhances the ability of BMP2 to stimulate chondrogenesis and cartilage specific gene expression, and reduces the concentration of BMP2 needed to stimulate chondrogenesis. Furthermore, HS stimulates BMP2-mediated phosphorylation of Smad1, Smad5, and Smad8, transcriptional mediators of BMP2 signaling, indicating that HS enhances the interaction of BMP2 with its receptors. Pretreatment of micromass cultures with heparitinase to degrade endogenous HSPGs also enhances the chondrogenic activity of BMP2, and reduces the concentration of BMP2 needed to promote chondrogenesis. Taken together these results indicate that exogenous HS or heparitinase enhance the chondrogenic activity of BMP2 by interfering with its interaction with endogenous HSPGs that would normally restrict its interaction with its receptors. Consistent with the possibility that HSPGs are negative modulators of BMP signaling during chondrogenesis, we have found that overexpression of syndecan-3, which is one of the major HSPGs normally expressed during chondrogenesis, greatly impairs the ability of BMP2 to promote cartilage differentiation. Furthermore, retroviral overexpression of syndecan-3 inhibits BMP2-mediated Smad phosphorylation in the regions of the cultures in which chondrogenesis is inhibited and in which ectopic syndecan-3 protein is highly expressed. These results indicate that syndecan-3 interferes with the interaction of BMP2 with its receptors, and that this interference results in an inhibition of chondrogenesis. Taken together these results indicate that HSPGs including syndecan-3 normally modulate the strength of BMP signaling during limb cartilage differentiation by limiting the effective concentration of BMP available for signaling.

Animals↗

PubMed2005

N-cadherin mediated distribution of beta-catenin alters MAP kinase and BMP-2 signaling on chondrogenesis-related gene expression.

We have examined the effect of calcium-dependent adhesion, mediated by N-cadherin, on cell signaling during chondrogenesis of multipotential embryonic mouse C3H10T1/2 cells. The activity of chondrogenic genes, type II collagen, aggrecan, and Sox9 were examined in monolayer (non-chondrogenic), and micromass (chondrogenic) cultures of parental C3H10T1/2 cells and altered C3H10T1/2 cell lines that express a dominant negative form of N-cadherin (delta390-T1/2) or overexpress normal N-cadherin (MNCD2-T1/2). Our findings show that missexpression or inhibition of N-cadherin in C3H10T1/2 cells results in temporal and spatial changes in expression of the chondrogenic genes Sox9, aggrecan, and collagen type II. We have also analyzed activity of the serum response factor (SRF), a nuclear target of MAP kinase signaling implicated in chondrogenesis. In semi-confluent monolayer cultures (minimum cell-cell contact) of C3H10T1/2, MNCD2-T1/2, or delta390-T1/2 cells, there was no significant change in the pattern of MAP kinase or bone morphogenetic protein-2 (BMP-2) regulation of SRF. However, in micromass cultures, the effect of MAP kinase and BMP-2 on SRF activity was proportional to the nuclear localization of beta-catenin, a Wnt stabilized cytoplasmic factor that can associate with lymphoid enhancer-binding factor (LEF) to serve as a transcription factor. Our findings suggest that the extent of adherens junction formation mediated by N-cadherin can modulate the potential Wnt-induced nuclear activity of beta-catenin.

Animals↗

PubMed2003

Progression of chondrogenesis in C3H10T1/2 cells is associated with prolonged and tight regulation of ERK1/2.

Close contact of mesenchymal cells in vivo and also in super dense micromass cultures in vitro results in cellular condensation and alteration of existing cellular signaling required for initiation and progression of chondrogenesis. To investigate chondrogenesis related changes in the activity of ubiquitous cell signaling mediated by mitogen-activated protein kinases (MAP kinase), we have compared the effect of cell seeding of pluripotent C3H10T1/2 mesenchymal cells as monolayers (non-chondrogenic culture) or high density micromass cultures (chondrogenic) on the regulation and phosphorylation state of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and also on regulation of ERK1/2 nuclear targets, namely, activation protein-1 (AP-1) and serum response factor (SRF). Increasing cell density resulted in reduced DNA binding as well as activity of AP-1. SRF activity, on the other hand, was up-regulated in confluent monolayer cultures but like AP-1 was inhibited in micromass cultures. Low levels of PD 98059 (5 microM), a specific inhibitor of ERK1/2, resulted in delayed induction of AP-1 and SRF activity whereas higher concentrations of this inhibitor (10-50 microM) conferred an opposite effect. Increasing concentrations of the PD 98059 inhibitor in long term monolayer or micromass cultures (2.5 day) resulted in differential regulation of c-Fos and c-Jun protein levels as well as total expression and phosphorylation levels of ERK1/2. PD 98059 treatment of C3H10T1/2 micromass cultures also resulted in up-regulation of type IIB collagen and Sox9 gene expression. While high expression of aggrecan and type IIB collagen genes were dependent on BMP-2 signaling, ERK inhibition of BMP-2 treated micromass cultures resulted in reduced activity of both genes. Our findings show that the activity of ERK1/2 in chondrogenic cultures of C3H10T1/2 cells is tightly controlled and can cross interact with other signaling activities mediated by BMP-2 to positively regulate chondrogensis.

Animals↗

PubMed2003

A simple, high-yield method for obtaining multipotential mesenchymal progenitor cells from trabecular bone.

In vitro cultures of primary, human trabecular bone-derived cells represent a useful system for investigation of the biology of osteoblasts. Our recent discovery of the multilineage mesenchymal differentiation potential of trabecular bone-derived cells suggests the potential application of these cells as mesenchymal progenitors for tissue repair and regeneration. Such applications are crucially dependent on efficient cell-isolation protocols to yield cells that optimally proliferate and differentiate. In this study, we describe a simple, high-yield procedure, requiring minimal culture expansion, for the isolation of mesenchymal progenitor cells from human trabecular bone. Moreover, these cells retain their ability to differentiate along multiple mesenchymal lineages through successive subculturing. Cell populations isolated and cultured as described here allow the efficient acquisition of a clinically significant number of cells, which may be used as the cell source for tissue-engineering applications.

Aged↗

PubMed2002

Cell density dependent regulation of AP-1 activity is important for chondrogenic differentiation of C3H10T1/2 mesenchymal cells.

The multipotential C3H10T1/2 mesenchymal cells undergo chondrogenic differentiation only when seeded as high-density micromass cultures, particularly upon treatment with bone morphogenetic protein-2 (BMP-2). The molecular mechanism(s) responsible for the cell density-dependent onset of cartilage-specific gene expression is presently unknown. Interestingly, a number of recent studies have indicated that activating protein-1 (AP-1), a well known downstream target of the mitogenic activated protein kinase (MAP kinase) signaling pathway, is a target of chondrogenic/osteogenic growth factors such as BMP-2, and plays a role in osteogenic gene regulation as well as in chondrogenic differentiation. The aim of this study is to examine the density-dependent alteration in the level and binding activity of AP-1 and its functional involvement in C3H10T1/2 mesenchymal chondrogenesis. To measure the activity of the AP-1 transcription factor, we generated a pool of stable C3H10T1/2 cell lines harboring a luciferase expression vector driven by a concatamer of an efficient AP-1 response element (AP1-10T1/2 cells). Luciferase activity of AP1-10T1/2 cultures was found to decrease sharply with increase in cell density, either as a function of culture time or initial cell seeding densities. In C3H10T1/2 micromass cultures undergoing chondrogenesis, AP-1 activity was further reduced and then maintained at a low, steady level for the entire 3-4 day culture period. AP-1 activity in micromass cultures was not significantly affected by BMP-2 treatment, but chondrogenesis was compromised upon competitive inhibition of AP-1 activity with a double-stranded AP-1 binding oligonucleotide. The level of AP-1 binding correlated with the activity of its response element but not with the levels of its leucine-zipper containing subunits, c-Jun and c-Fos. These findings suggest that a cell density-dependent, low but steady level of AP-1 binding and activity is required for promoting the chondrogenic potential of C3H10T1/2 cells.

3T3 Cells↗

PubMed2002

Distribution and possible function of an adrenomedullin-like peptide in the developing chick limb bud.

Adrenomedullin (AM) is a multifunctional peptide that exhibits discrete domains of expression during mouse embryogenesis consistent with a role in regulating growth and differentiation during morphogenesis. Here we report that AM immunoreactivity is present at high levels throughout the apical ectodermal ridge (AER) of the chick limb bud as the AER is directing the outgrowth and patterning of underlying limb mesoderm. Immunostaining is particularly strong along the surfaces of the contiguous cells of the AER. AM immunoreactivity attenuates as the AER regresses and is absent from the distal apical ectoderm of stage 20 limbless mutant limb buds which fail to develop an AER. To explore the possible role of AM in AER activity, we examined the effect of exogenous AM and an AM inhibitor on the in vitro morphogenesis of limb mesoderm, cultured in the presence and absence of the AER. Although exogenous AM cannot substitute for the AER in promoting outgrowth of limb mesoderm in vitro, a specific AM antagonist, AM(22-52), impairs the outgrowth and proliferation of limb mesoderm cultured in the presence of the AER. This is consistent with the possibility that inhibition of endogenous AM activity in the AER impairs the ability of the AER to promote limb morphogenesis. Taken together, these studies suggest that an AM-like molecule may function in an autocrine fashion to regulate some aspect of AER activity.

Adrenomedullin↗