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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: 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.Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces.

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: 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.Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces.

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