Bennett K L, Jackson D G, Simon J C, Tanczos E, Peach R, Modrell B, Stamenkovic I, Plowman G, Aruffo A
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
J Cell Biol. 1995 Feb;128(4):687-98. doi: 10.1083/jcb.128.4.687.
Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS-modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS-modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS-modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS-binding growth factors to leukocytes on the vascular cell wall.
CD44的糖胺聚糖修饰异构体与炎症部位的生长因子呈递有关。在本研究中，我们发现表达含有可变剪接外显子V3的CD44异构体的COS细胞转染子被硫酸乙酰肝素（HS）修饰。与三种HS结合生长因子，即碱性成纤维细胞生长因子（b-FGF）、肝素结合表皮生长因子（HB-EGF）和双调蛋白的结合研究表明，HS修饰的CD44异构体能够结合b-FGF和HB-EGF，但不能结合双调蛋白。用硫酸乙酰肝素酶预处理蛋白质或用游离肝素阻断可消除b-FGF和HB-EGF与HS修饰的CD44的结合。从表达含V3的CD44异构体的角质形成细胞中免疫沉淀的HS修饰的CD44也能结合b-FGF。我们研究了HS修饰的CD44异构体是否由活化的内皮细胞表达，在炎症反应期间它们可能将HS结合生长因子呈递给白细胞。PCR和抗体结合研究表明，活化的培养内皮细胞仅表达不包含任何可变剪接外显子（包括V3）的CD44H异构体。用针对可变剪接外显子编码的CD44细胞外结构域的抗体进行的免疫组织学研究表明，炎症皮肤组织切片中的血管内皮细胞不表达CD44剪接变体。在相同标本中发现角质形成细胞、单核细胞和树突状细胞表达可变剪接的CD44。35SO4(-2)标记实验表明，活化的培养内皮细胞不表达可检测水平的硫酸软骨素或HS修饰的CD44。我们的结果表明，表达V3的CD44异构体的功能之一是结合并呈递一部分HS结合蛋白。此外，HS修饰的CD44可能参与炎症皮肤中角质形成细胞对HS结合蛋白的呈递。然而，我们的数据表明，CD44不太可能是主要参与在血管细胞壁上向白细胞呈递HS结合生长因子的蛋白聚糖。
