Fisher Melanie C, Li Yingcui, Seghatoleslami M Reza, Dealy Caroline N, Kosher Robert A
Center for Regenerative Medicine and Skeletal Development, MC3705, Department of Oral Rehabilitation, Biomaterials, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.
Matrix Biol. 2006 Jan;25(1):27-39. doi: 10.1016/j.matbio.2005.07.008. Epub 2005 Oct 13.
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.
骨形态发生蛋白（BMPs）参与肢体发育的多个方面，包括软骨分化的调节。几种BMPs与肝素紧密结合，最近发现细胞表面或细胞外基质中的硫酸乙酰肝素蛋白聚糖（HSPGs）在一些发育系统中作为BMP信号传导的调节剂。在这里，我们通过评估外源性硫酸乙酰肝素（HS）、肝素酶处理以及HSPG syndecan-3的过表达对BMP2调节微团培养中肢体间充质细胞软骨分化能力的影响，探讨了HSPGs在肢体软骨形成过程中调节BMP活性的作用。外源性HS显著增强BMP2刺激软骨形成和软骨特异性基因表达的能力，并降低刺激软骨形成所需的BMP2浓度。此外，HS刺激BMP2介导的Smad1、Smad5和Smad8的磷酸化，Smad1、Smad5和Smad8是BMP2信号传导的转录介质，表明HS增强了BMP2与其受体的相互作用。用肝素酶预处理微团培养物以降解内源性HSPGs也增强了BMP2的软骨形成活性，并降低了促进软骨形成所需的BMP2浓度。综合这些结果表明，外源性HS或肝素酶通过干扰BMP2与内源性HSPGs的相互作用来增强BMP2的软骨形成活性，而内源性HSPGs通常会限制BMP2与其受体的相互作用。与HSPGs在软骨形成过程中是BMP信号传导的负调节剂这一可能性一致，我们发现syndecan-3（软骨形成过程中正常表达的主要HSPGs之一）的过表达极大地损害了BMP2促进软骨分化的能力。此外，syndecan-3的逆转录病毒过表达在软骨形成受到抑制且异位syndecan-3蛋白高度表达的培养区域中抑制BMP2介导的Smad磷酸化。这些结果表明syndecan-3干扰BMP2与其受体的相互作用，并且这种干扰导致软骨形成的抑制。综合这些结果表明，包括syndecan-3在内的HSPGs通常通过限制可用于信号传导的BMP的有效浓度来调节肢体软骨分化过程中BMP信号传导的强度。
