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Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts.

Li S, Liquari P, McKee KK, Harrison D, Patel R, Lee S, Yurchenco PD - J. Cell Biol. (2005)

Bottom Line: This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly.Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces.Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.

ABSTRACT
Endoneurial laminins (Lms), beta1-integrins, and dystroglycan (DG) are important for Schwann cell (SC) ensheathment and myelination of axons. We now show that SC expression of galactosyl-sulfatide, a Lm-binding glycolipid, precedes that of Lms in developing nerves. This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly. Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces. Assembly is characterized by coalescence of sulfatide, DG, and c-Src into a Lm-associated complex; by DG-dependent recruitment of utrophin and Src activation; and by integrin-dependent focal adhesion kinase phosphorylation. Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.

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Tyrosine phosphorylation of c-Src and caveolin-1 in sulfatide-loaded fibroblasts. (a) MEFs were loaded with gal-sulfatide and treated with 10 μg/ml Lm-1. Equal protein loads of cell lysates were analyzed in immunoblots. Transient Src activation (PY416) was detected within 30 min after Lm-1 treatment. (bottom left) Ratio of Src-PY416/total Src. (b) Lm-1 does not induce Src phosphorylation in fibroblasts in the absence of sulfatide loading. Fibroblasts with or without sulfatide loading were incubated with 10 μg/ml Lm-1 for 1 h. Cell lysates were immunoblotted with either c-Src-Py416 or c-Src–specific antibodies. (c) αDG antibody and Lm-1 fragment E3 inhibit Src phosphorylation in sulfatide-loaded fibroblasts treated with Lm-1. Gal-sulfatide–loaded fibroblasts were treated with 10 μg/ml Lm-1 for 1 h in the presence of either 100 μg/ml BSA,100 μg/ml E3, 250 μg/ml E8, 10 μg/ml mouse IgM, 10 μg/ml IIH6, or 10 μg/ml of β1-integrin antibody Ha2/5; lysed; and immunoblotted for pSrc and c-Src. (d) DG expression is required for Lm induction of Src activation in sulfatide-loaded fibroblasts. Fibroblasts derived from wild-type or DG- embryonic stem cells treated with gal-sulfatide were incubated with 10 μg/ml Lm-1 for 1 h and analyzed for pSrc and total Src. (e) Ablation of the β1-integrin gene does not prevent Lm-1–induced Src phosphorylation in sulfatide-loaded fibroblasts. β1-integrin–deficient fibroblasts (GD25) and β1-integrin–transduced GD25 control cells were treated the same as described in d, with lysates analyzed for pSrc and total Src. (f and g) Lm-1 assembly on sulfatide-loaded fibroblast surfaces does not require DG or β1-integrin. DG- (f) and β1-integrin– (g) fibroblasts, loaded with gal-sulfatide and incubated with 10 μg/ml Lm-1 for 1 h, were fixed and immunostained for Lm α1. (h) Caveolin-1 phosphorylation is induced by Lm-1 in sulfatide-treated embryonic lung fibroblasts. Fibroblasts were treated the same as described in panel a, and analyzed for Py14-caveolin-1 (Cav-1). The densitometry plot (caveolin-1-Py14/total caveolin-1) is also shown. (i) Src inhibition decreases Lm-induced caveolin-1 phosphorylation. Sulfatide-loaded fibroblasts were treated with Lm-1 plus Src kinase inhibitor PP2 (2 μM) or SU6656 (2 μM) for 1 h. Cell lysates were analyzed in immunoblots for caveolin-1-Py14 (Cav-1-Py14) or total caveolin-1 (Cav-1).
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fig7: Tyrosine phosphorylation of c-Src and caveolin-1 in sulfatide-loaded fibroblasts. (a) MEFs were loaded with gal-sulfatide and treated with 10 μg/ml Lm-1. Equal protein loads of cell lysates were analyzed in immunoblots. Transient Src activation (PY416) was detected within 30 min after Lm-1 treatment. (bottom left) Ratio of Src-PY416/total Src. (b) Lm-1 does not induce Src phosphorylation in fibroblasts in the absence of sulfatide loading. Fibroblasts with or without sulfatide loading were incubated with 10 μg/ml Lm-1 for 1 h. Cell lysates were immunoblotted with either c-Src-Py416 or c-Src–specific antibodies. (c) αDG antibody and Lm-1 fragment E3 inhibit Src phosphorylation in sulfatide-loaded fibroblasts treated with Lm-1. Gal-sulfatide–loaded fibroblasts were treated with 10 μg/ml Lm-1 for 1 h in the presence of either 100 μg/ml BSA,100 μg/ml E3, 250 μg/ml E8, 10 μg/ml mouse IgM, 10 μg/ml IIH6, or 10 μg/ml of β1-integrin antibody Ha2/5; lysed; and immunoblotted for pSrc and c-Src. (d) DG expression is required for Lm induction of Src activation in sulfatide-loaded fibroblasts. Fibroblasts derived from wild-type or DG- embryonic stem cells treated with gal-sulfatide were incubated with 10 μg/ml Lm-1 for 1 h and analyzed for pSrc and total Src. (e) Ablation of the β1-integrin gene does not prevent Lm-1–induced Src phosphorylation in sulfatide-loaded fibroblasts. β1-integrin–deficient fibroblasts (GD25) and β1-integrin–transduced GD25 control cells were treated the same as described in d, with lysates analyzed for pSrc and total Src. (f and g) Lm-1 assembly on sulfatide-loaded fibroblast surfaces does not require DG or β1-integrin. DG- (f) and β1-integrin– (g) fibroblasts, loaded with gal-sulfatide and incubated with 10 μg/ml Lm-1 for 1 h, were fixed and immunostained for Lm α1. (h) Caveolin-1 phosphorylation is induced by Lm-1 in sulfatide-treated embryonic lung fibroblasts. Fibroblasts were treated the same as described in panel a, and analyzed for Py14-caveolin-1 (Cav-1). The densitometry plot (caveolin-1-Py14/total caveolin-1) is also shown. (i) Src inhibition decreases Lm-induced caveolin-1 phosphorylation. Sulfatide-loaded fibroblasts were treated with Lm-1 plus Src kinase inhibitor PP2 (2 μM) or SU6656 (2 μM) for 1 h. Cell lysates were analyzed in immunoblots for caveolin-1-Py14 (Cav-1-Py14) or total caveolin-1 (Cav-1).

Mentions: Sulfatide-treated fibroblasts were then evaluated for Src tyrosine phosphorylation (Fig. 7) in response to Lm-1. Lm-1 induced a similar transient activation of c-Src in sulfatide-loaded cells that was maximal at 1 h (Fig. 7 a). This was not observed if the fibroblasts were treated with Lm but not loaded with sulfatide (Fig. 7 b). Src phosphorylation was blocked partially by fragment E3 (as seen with SCs) and fully by the DG antibody IIH6, but not with antibody Ha2/5 to β1-integrin or by Lm fragment E8 that possesses the α6β1-integrin–binding locus (Fig. 7 c). To further examine the role of DG and β1-integrin in Src activation, we evaluated cultures of fibroblasts isolated from differentiated mouse embryonic stem (ES) cells that were genetically for DG or for β1-integrin, and compared these with fibroblasts derived from wild-type ES cells or ones that were transfected with a construct to enable expression of β1-integrin (β1AGD25 cells; Wennerberg et al., 1996). The cells were cultured on plastic, loaded with gal-sulfatide, and incubated in the presence of Lm-1. Src activation was observed in response to Lm-1 in the wild-type, but not the DG-, fibroblasts (Fig. 7 d). In contrast, Lm-1 stimulated an increased Src activation in both control and β1-integrin– fibroblasts, although at an approximately twofold higher level in the control cells (Fig. 7 e). Because β1-integrin did not colocalize with Lm under these conditions, and because β1-integrin–blocking antibody Ha2/5 did not inhibit Lm-induced Src phosphorylation, it was thought likely that Src activation was primarily dependent on DG and that the integrin contribution was independent of Lm assembly. Lm-1 accumulated on both DG- and β1-integrin– fibroblasts (Fig. 7, f and g). Finally, caveolin-1 became transiently phosphorylated at tyrosine-14 with a similar time course in fibroblasts, unlike SCs (Fig. 7 h). Inhibition of Src kinases with two structurally different inhibitors (PP2 and SU6656) inhibited caveolin-1 phosphorylation (Fig. 7 i), suggesting caveolin-1 was a downstream target of the Lm-activated Src.


Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts.

Li S, Liquari P, McKee KK, Harrison D, Patel R, Lee S, Yurchenco PD - J. Cell Biol. (2005)

Tyrosine phosphorylation of c-Src and caveolin-1 in sulfatide-loaded fibroblasts. (a) MEFs were loaded with gal-sulfatide and treated with 10 μg/ml Lm-1. Equal protein loads of cell lysates were analyzed in immunoblots. Transient Src activation (PY416) was detected within 30 min after Lm-1 treatment. (bottom left) Ratio of Src-PY416/total Src. (b) Lm-1 does not induce Src phosphorylation in fibroblasts in the absence of sulfatide loading. Fibroblasts with or without sulfatide loading were incubated with 10 μg/ml Lm-1 for 1 h. Cell lysates were immunoblotted with either c-Src-Py416 or c-Src–specific antibodies. (c) αDG antibody and Lm-1 fragment E3 inhibit Src phosphorylation in sulfatide-loaded fibroblasts treated with Lm-1. Gal-sulfatide–loaded fibroblasts were treated with 10 μg/ml Lm-1 for 1 h in the presence of either 100 μg/ml BSA,100 μg/ml E3, 250 μg/ml E8, 10 μg/ml mouse IgM, 10 μg/ml IIH6, or 10 μg/ml of β1-integrin antibody Ha2/5; lysed; and immunoblotted for pSrc and c-Src. (d) DG expression is required for Lm induction of Src activation in sulfatide-loaded fibroblasts. Fibroblasts derived from wild-type or DG- embryonic stem cells treated with gal-sulfatide were incubated with 10 μg/ml Lm-1 for 1 h and analyzed for pSrc and total Src. (e) Ablation of the β1-integrin gene does not prevent Lm-1–induced Src phosphorylation in sulfatide-loaded fibroblasts. β1-integrin–deficient fibroblasts (GD25) and β1-integrin–transduced GD25 control cells were treated the same as described in d, with lysates analyzed for pSrc and total Src. (f and g) Lm-1 assembly on sulfatide-loaded fibroblast surfaces does not require DG or β1-integrin. DG- (f) and β1-integrin– (g) fibroblasts, loaded with gal-sulfatide and incubated with 10 μg/ml Lm-1 for 1 h, were fixed and immunostained for Lm α1. (h) Caveolin-1 phosphorylation is induced by Lm-1 in sulfatide-treated embryonic lung fibroblasts. Fibroblasts were treated the same as described in panel a, and analyzed for Py14-caveolin-1 (Cav-1). The densitometry plot (caveolin-1-Py14/total caveolin-1) is also shown. (i) Src inhibition decreases Lm-induced caveolin-1 phosphorylation. Sulfatide-loaded fibroblasts were treated with Lm-1 plus Src kinase inhibitor PP2 (2 μM) or SU6656 (2 μM) for 1 h. Cell lysates were analyzed in immunoblots for caveolin-1-Py14 (Cav-1-Py14) or total caveolin-1 (Cav-1).
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fig7: Tyrosine phosphorylation of c-Src and caveolin-1 in sulfatide-loaded fibroblasts. (a) MEFs were loaded with gal-sulfatide and treated with 10 μg/ml Lm-1. Equal protein loads of cell lysates were analyzed in immunoblots. Transient Src activation (PY416) was detected within 30 min after Lm-1 treatment. (bottom left) Ratio of Src-PY416/total Src. (b) Lm-1 does not induce Src phosphorylation in fibroblasts in the absence of sulfatide loading. Fibroblasts with or without sulfatide loading were incubated with 10 μg/ml Lm-1 for 1 h. Cell lysates were immunoblotted with either c-Src-Py416 or c-Src–specific antibodies. (c) αDG antibody and Lm-1 fragment E3 inhibit Src phosphorylation in sulfatide-loaded fibroblasts treated with Lm-1. Gal-sulfatide–loaded fibroblasts were treated with 10 μg/ml Lm-1 for 1 h in the presence of either 100 μg/ml BSA,100 μg/ml E3, 250 μg/ml E8, 10 μg/ml mouse IgM, 10 μg/ml IIH6, or 10 μg/ml of β1-integrin antibody Ha2/5; lysed; and immunoblotted for pSrc and c-Src. (d) DG expression is required for Lm induction of Src activation in sulfatide-loaded fibroblasts. Fibroblasts derived from wild-type or DG- embryonic stem cells treated with gal-sulfatide were incubated with 10 μg/ml Lm-1 for 1 h and analyzed for pSrc and total Src. (e) Ablation of the β1-integrin gene does not prevent Lm-1–induced Src phosphorylation in sulfatide-loaded fibroblasts. β1-integrin–deficient fibroblasts (GD25) and β1-integrin–transduced GD25 control cells were treated the same as described in d, with lysates analyzed for pSrc and total Src. (f and g) Lm-1 assembly on sulfatide-loaded fibroblast surfaces does not require DG or β1-integrin. DG- (f) and β1-integrin– (g) fibroblasts, loaded with gal-sulfatide and incubated with 10 μg/ml Lm-1 for 1 h, were fixed and immunostained for Lm α1. (h) Caveolin-1 phosphorylation is induced by Lm-1 in sulfatide-treated embryonic lung fibroblasts. Fibroblasts were treated the same as described in panel a, and analyzed for Py14-caveolin-1 (Cav-1). The densitometry plot (caveolin-1-Py14/total caveolin-1) is also shown. (i) Src inhibition decreases Lm-induced caveolin-1 phosphorylation. Sulfatide-loaded fibroblasts were treated with Lm-1 plus Src kinase inhibitor PP2 (2 μM) or SU6656 (2 μM) for 1 h. Cell lysates were analyzed in immunoblots for caveolin-1-Py14 (Cav-1-Py14) or total caveolin-1 (Cav-1).
Mentions: Sulfatide-treated fibroblasts were then evaluated for Src tyrosine phosphorylation (Fig. 7) in response to Lm-1. Lm-1 induced a similar transient activation of c-Src in sulfatide-loaded cells that was maximal at 1 h (Fig. 7 a). This was not observed if the fibroblasts were treated with Lm but not loaded with sulfatide (Fig. 7 b). Src phosphorylation was blocked partially by fragment E3 (as seen with SCs) and fully by the DG antibody IIH6, but not with antibody Ha2/5 to β1-integrin or by Lm fragment E8 that possesses the α6β1-integrin–binding locus (Fig. 7 c). To further examine the role of DG and β1-integrin in Src activation, we evaluated cultures of fibroblasts isolated from differentiated mouse embryonic stem (ES) cells that were genetically for DG or for β1-integrin, and compared these with fibroblasts derived from wild-type ES cells or ones that were transfected with a construct to enable expression of β1-integrin (β1AGD25 cells; Wennerberg et al., 1996). The cells were cultured on plastic, loaded with gal-sulfatide, and incubated in the presence of Lm-1. Src activation was observed in response to Lm-1 in the wild-type, but not the DG-, fibroblasts (Fig. 7 d). In contrast, Lm-1 stimulated an increased Src activation in both control and β1-integrin– fibroblasts, although at an approximately twofold higher level in the control cells (Fig. 7 e). Because β1-integrin did not colocalize with Lm under these conditions, and because β1-integrin–blocking antibody Ha2/5 did not inhibit Lm-induced Src phosphorylation, it was thought likely that Src activation was primarily dependent on DG and that the integrin contribution was independent of Lm assembly. Lm-1 accumulated on both DG- and β1-integrin– fibroblasts (Fig. 7, f and g). Finally, caveolin-1 became transiently phosphorylated at tyrosine-14 with a similar time course in fibroblasts, unlike SCs (Fig. 7 h). Inhibition of Src kinases with two structurally different inhibitors (PP2 and SU6656) inhibited caveolin-1 phosphorylation (Fig. 7 i), suggesting caveolin-1 was a downstream target of the Lm-activated Src.

Bottom Line: This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly.Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces.Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.

ABSTRACT
Endoneurial laminins (Lms), beta1-integrins, and dystroglycan (DG) are important for Schwann cell (SC) ensheathment and myelination of axons. We now show that SC expression of galactosyl-sulfatide, a Lm-binding glycolipid, precedes that of Lms in developing nerves. This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly. Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces. Assembly is characterized by coalescence of sulfatide, DG, and c-Src into a Lm-associated complex; by DG-dependent recruitment of utrophin and Src activation; and by integrin-dependent focal adhesion kinase phosphorylation. Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.

Show MeSH
Related in: MedlinePlus