Limits...
Cell adhesion and signaling on the fibronectin 1st type III repeat; requisite roles for cell surface proteoglycans and integrins.

Mercurius KO, Morla AO - BMC Cell Biol. (2001)

Bottom Line: Cell adhesion to III1-C results in robust ERK1/2 activation that is blocked by integrin-blocking agents.Moreover, heparitinase treatment, but not chondroitinase treatment of RASMCs results in reduced cell adhesion and ERK1/2 activation.The results suggest that the 1st type III repeat of fibronectin contains a previously unrecognized cell adhesion domain that stimulates robust ERK1/2 activation in RASMCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, MC 1089 Committee on Cancer Biology, University of Chicago, S Maryland Ave, Chicago, IL, USA. kmercuri@midway.uchicago.edu

ABSTRACT

Background: The first type III repeat of fibronectin is known to be involved in fibronectin matrix assembly, and recombinant proteins from this type III repeat can inhibit cell proliferation, tumor metastasis and angiogenesis. We have analyzed the way rat aortic smooth muscle cells (RASMCs) interact with a recombinant protein encompassing a C-terminal portion of the first type III repeat of fibronectin (protein III1-C).

Results: Cells are able to adhere to and spread on III1-C coated on a dish. Both beta1 integrins and cell surface heparan sulfate proteoglycans serve as receptors for III1-C. For example, cell attachment to III1-C is partially inhibited by agents that block beta1 integrins or by heparin. Complete inhibition of cell attachment is seen only when integrin blocking agents are combined with heparin. Affinity chromatography revealed the binding of proteins that likely represent the integrin beta1 and alpha5 submits to a III1-C column. Cell adhesion to III1-C results in robust ERK1/2 activation that is blocked by integrin-blocking agents. In addition, cell adhesion to III1-C and ERK1/2 activation by III1-C are both inhibited by heparan sulfate but not by chondroitin sulfate. Moreover, heparitinase treatment, but not chondroitinase treatment of RASMCs results in reduced cell adhesion and ERK1/2 activation. Affinity chromatography experiments demonstrated that 35SO4-labeled cell surface heparan sulfate proteoglycans bound specifically to III1-C.

Conclusions: The results suggest that the 1st type III repeat of fibronectin contains a previously unrecognized cell adhesion domain that stimulates robust ERK1/2 activation in RASMCs. Cells interact with this domain through cell surface heparan sulfate proteoglycans and integrins, and both classes of receptors are required for optimal cell adhesion and ERK1/2 activation.

Show MeSH

Related in: MedlinePlus

Affinity chromatography HSPGs on III1-C Sepharose. RASMCs were collected by trypsinization and lysed in NP40 buffer. The lysate was then treated either without (-) or with (+) 0.1 u/ml heparitinase for 1 hr at 37°C. Heparitinase treated samples were then applied to either III1-C Sepharose (lanes marked C) or III 11-C Sepharose (lanes marked 11C). One sample was applied to a III1-C Sepharose column in the presence of 0.5 mg/ml heparin (lanes marked C•H). The flow through fractions were collected (FT lanes), the columns were washed extensively, then the Sepharose beads were collected and boiled in SDS-PAGE sample buffer (Bound lanes). Samples were analyzed by immunoblotting with the 3G10 antibody. Antibody 3G10 recognizes the uronate stubs that remain associated with core proteins after heparitinase digestion. Thus, after heparitinase digestion 3G10 shows the sizes of HSPG core proteins. Note that the major HSPGs have core protein sizes of 46 kDa and 70 kDa, and both of these HSPGs bind to the III1-C column but not the III 11-C column. The experiment was performed twice with similar results both times.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC57736&req=5

Figure 10: Affinity chromatography HSPGs on III1-C Sepharose. RASMCs were collected by trypsinization and lysed in NP40 buffer. The lysate was then treated either without (-) or with (+) 0.1 u/ml heparitinase for 1 hr at 37°C. Heparitinase treated samples were then applied to either III1-C Sepharose (lanes marked C) or III 11-C Sepharose (lanes marked 11C). One sample was applied to a III1-C Sepharose column in the presence of 0.5 mg/ml heparin (lanes marked C•H). The flow through fractions were collected (FT lanes), the columns were washed extensively, then the Sepharose beads were collected and boiled in SDS-PAGE sample buffer (Bound lanes). Samples were analyzed by immunoblotting with the 3G10 antibody. Antibody 3G10 recognizes the uronate stubs that remain associated with core proteins after heparitinase digestion. Thus, after heparitinase digestion 3G10 shows the sizes of HSPG core proteins. Note that the major HSPGs have core protein sizes of 46 kDa and 70 kDa, and both of these HSPGs bind to the III1-C column but not the III 11-C column. The experiment was performed twice with similar results both times.

Mentions: RASMC HSPGs were analyzed further by immunoblotting with the monoclonal antibody 3G10 which specifically recognizes desaturated uronates on the heparan sulfate stubs that remain associated with core proteins after heparitinase digestion [37]. Complete digestion of RASMC lysates with heparitinase reveals major bands at 46 kDa and 70 kDa, indicating that these are the sizes of the major HSPG core proteins (not shown). RASMC lysates were subjected to partial heparitinase treatment (full digestion was not performed in order to allow some GAGs to remain associated with the core proteins) then applied to either III1-C or III 11-C Sepharose. As shown in Fig. 10, most of the 46 kDa and 70 kDa HSPGs bound to III1-C Sepharose but not to III 11-C Sepharose. In addition, the presence of heparin in the lysate inhibited the 46 kDa and 70 kDa HSPGs from binding to the III1-C column (Fig. 10, compare C and C•H lanes). The inhibition of 46 kDa and 70 kDa binding to III1-C by heparin suggests that these HSPGs may serve as receptors for III1-C in cell adhesion and ERK1/2 signaling.


Cell adhesion and signaling on the fibronectin 1st type III repeat; requisite roles for cell surface proteoglycans and integrins.

Mercurius KO, Morla AO - BMC Cell Biol. (2001)

Affinity chromatography HSPGs on III1-C Sepharose. RASMCs were collected by trypsinization and lysed in NP40 buffer. The lysate was then treated either without (-) or with (+) 0.1 u/ml heparitinase for 1 hr at 37°C. Heparitinase treated samples were then applied to either III1-C Sepharose (lanes marked C) or III 11-C Sepharose (lanes marked 11C). One sample was applied to a III1-C Sepharose column in the presence of 0.5 mg/ml heparin (lanes marked C•H). The flow through fractions were collected (FT lanes), the columns were washed extensively, then the Sepharose beads were collected and boiled in SDS-PAGE sample buffer (Bound lanes). Samples were analyzed by immunoblotting with the 3G10 antibody. Antibody 3G10 recognizes the uronate stubs that remain associated with core proteins after heparitinase digestion. Thus, after heparitinase digestion 3G10 shows the sizes of HSPG core proteins. Note that the major HSPGs have core protein sizes of 46 kDa and 70 kDa, and both of these HSPGs bind to the III1-C column but not the III 11-C column. The experiment was performed twice with similar results both times.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC57736&req=5

Figure 10: Affinity chromatography HSPGs on III1-C Sepharose. RASMCs were collected by trypsinization and lysed in NP40 buffer. The lysate was then treated either without (-) or with (+) 0.1 u/ml heparitinase for 1 hr at 37°C. Heparitinase treated samples were then applied to either III1-C Sepharose (lanes marked C) or III 11-C Sepharose (lanes marked 11C). One sample was applied to a III1-C Sepharose column in the presence of 0.5 mg/ml heparin (lanes marked C•H). The flow through fractions were collected (FT lanes), the columns were washed extensively, then the Sepharose beads were collected and boiled in SDS-PAGE sample buffer (Bound lanes). Samples were analyzed by immunoblotting with the 3G10 antibody. Antibody 3G10 recognizes the uronate stubs that remain associated with core proteins after heparitinase digestion. Thus, after heparitinase digestion 3G10 shows the sizes of HSPG core proteins. Note that the major HSPGs have core protein sizes of 46 kDa and 70 kDa, and both of these HSPGs bind to the III1-C column but not the III 11-C column. The experiment was performed twice with similar results both times.
Mentions: RASMC HSPGs were analyzed further by immunoblotting with the monoclonal antibody 3G10 which specifically recognizes desaturated uronates on the heparan sulfate stubs that remain associated with core proteins after heparitinase digestion [37]. Complete digestion of RASMC lysates with heparitinase reveals major bands at 46 kDa and 70 kDa, indicating that these are the sizes of the major HSPG core proteins (not shown). RASMC lysates were subjected to partial heparitinase treatment (full digestion was not performed in order to allow some GAGs to remain associated with the core proteins) then applied to either III1-C or III 11-C Sepharose. As shown in Fig. 10, most of the 46 kDa and 70 kDa HSPGs bound to III1-C Sepharose but not to III 11-C Sepharose. In addition, the presence of heparin in the lysate inhibited the 46 kDa and 70 kDa HSPGs from binding to the III1-C column (Fig. 10, compare C and C•H lanes). The inhibition of 46 kDa and 70 kDa binding to III1-C by heparin suggests that these HSPGs may serve as receptors for III1-C in cell adhesion and ERK1/2 signaling.

Bottom Line: Cell adhesion to III1-C results in robust ERK1/2 activation that is blocked by integrin-blocking agents.Moreover, heparitinase treatment, but not chondroitinase treatment of RASMCs results in reduced cell adhesion and ERK1/2 activation.The results suggest that the 1st type III repeat of fibronectin contains a previously unrecognized cell adhesion domain that stimulates robust ERK1/2 activation in RASMCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, MC 1089 Committee on Cancer Biology, University of Chicago, S Maryland Ave, Chicago, IL, USA. kmercuri@midway.uchicago.edu

ABSTRACT

Background: The first type III repeat of fibronectin is known to be involved in fibronectin matrix assembly, and recombinant proteins from this type III repeat can inhibit cell proliferation, tumor metastasis and angiogenesis. We have analyzed the way rat aortic smooth muscle cells (RASMCs) interact with a recombinant protein encompassing a C-terminal portion of the first type III repeat of fibronectin (protein III1-C).

Results: Cells are able to adhere to and spread on III1-C coated on a dish. Both beta1 integrins and cell surface heparan sulfate proteoglycans serve as receptors for III1-C. For example, cell attachment to III1-C is partially inhibited by agents that block beta1 integrins or by heparin. Complete inhibition of cell attachment is seen only when integrin blocking agents are combined with heparin. Affinity chromatography revealed the binding of proteins that likely represent the integrin beta1 and alpha5 submits to a III1-C column. Cell adhesion to III1-C results in robust ERK1/2 activation that is blocked by integrin-blocking agents. In addition, cell adhesion to III1-C and ERK1/2 activation by III1-C are both inhibited by heparan sulfate but not by chondroitin sulfate. Moreover, heparitinase treatment, but not chondroitinase treatment of RASMCs results in reduced cell adhesion and ERK1/2 activation. Affinity chromatography experiments demonstrated that 35SO4-labeled cell surface heparan sulfate proteoglycans bound specifically to III1-C.

Conclusions: The results suggest that the 1st type III repeat of fibronectin contains a previously unrecognized cell adhesion domain that stimulates robust ERK1/2 activation in RASMCs. Cells interact with this domain through cell surface heparan sulfate proteoglycans and integrins, and both classes of receptors are required for optimal cell adhesion and ERK1/2 activation.

Show MeSH
Related in: MedlinePlus