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The Rac activator Tiam1 is required for (alpha)3(beta)1-mediated laminin-5 deposition, cell spreading, and cell migration.

Hamelers IH, Olivo C, Mertens AE, Pegtel DM, van der Kammen RA, Sonnenberg A, Collard JG - J. Cell Biol. (2005)

Bottom Line: Both Tiam1 and V12Rac1 can rescue the defects of Tiam1-/- keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation.Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin.Our studies indicate that Tiam1 is a key molecule in alpha3beta1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
The Rho-like guanosine triphosphatase Rac1 regulates various signaling pathways, including integrin-mediated adhesion and migration of cells. However, the mechanisms by which integrins signal toward Rac are poorly understood. We show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis 1) is required for the integrin-mediated laminin (LN)-5 deposition, spreading, and migration of keratinocytes. In contrast to wild-type keratinocytes, Tiam1-deficient (Tiam1-/-) keratinocytes are unable to adhere to and spread on a glass substrate because they are unable to deposit their own LN5 substrate. Both Tiam1 and V12Rac1 can rescue the defects of Tiam1-/- keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation. Tiam1-/- cells are unable to activate Rac upon alpha3beta1-mediated adhesion to an exogenous LN5 substrate. Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin. Our studies indicate that Tiam1 is a key molecule in alpha3beta1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.

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Production and secretion of LN5 by WT and Tiam1−/− keratinocytes. (A) Suspended WT and Tiam1−/− keratinocytes were seeded on a glass surface or on a Col IV–coated surface. After 30 or 180 min, attached cells were detached with 10 mM EDTA and secreted LN5 was detached from the surface with SDS–sample buffer. As a control, suspended cells were lysed. The samples were subjected to Western blotting and probed for LN5 (γ2 chain). (B) The amounts of secreted LN5 (γ2 chain) levels in the Western blot shown in A were quantified relative to levels in the 180-min WT sample (=100%) with Image J. (C) Suspended WT, Tiam1−/−, and Tiam1−/− keratinocytes stably expressing full-length Tiam1 or RacV12 were seeded on a Col IV–coated surface. A sample of the cells in suspension was used for mRNA isolation. After 45 min, adherent cells were detached from the surface and lysed for mRNA isolation. An RT-PCR reaction was performed to determine the amount of LN5 γ2 chain mRNA present in the samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control. The histogram represents the increase in LN5 mRNA levels in both WT and Tiam1−/− cells determined in two independent experiments. Error bars represent the SD. (D) Cells were seeded on Col IV–coated coverslips for 16 h, fixed, and stained for plectin, the integrin β4 subunit, and the LN5 γ2 chain. Note the accumulation of LN5-containing vesicles in the Tiam1−/− cells. Bars, 20 μm.
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fig8: Production and secretion of LN5 by WT and Tiam1−/− keratinocytes. (A) Suspended WT and Tiam1−/− keratinocytes were seeded on a glass surface or on a Col IV–coated surface. After 30 or 180 min, attached cells were detached with 10 mM EDTA and secreted LN5 was detached from the surface with SDS–sample buffer. As a control, suspended cells were lysed. The samples were subjected to Western blotting and probed for LN5 (γ2 chain). (B) The amounts of secreted LN5 (γ2 chain) levels in the Western blot shown in A were quantified relative to levels in the 180-min WT sample (=100%) with Image J. (C) Suspended WT, Tiam1−/−, and Tiam1−/− keratinocytes stably expressing full-length Tiam1 or RacV12 were seeded on a Col IV–coated surface. A sample of the cells in suspension was used for mRNA isolation. After 45 min, adherent cells were detached from the surface and lysed for mRNA isolation. An RT-PCR reaction was performed to determine the amount of LN5 γ2 chain mRNA present in the samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control. The histogram represents the increase in LN5 mRNA levels in both WT and Tiam1−/− cells determined in two independent experiments. Error bars represent the SD. (D) Cells were seeded on Col IV–coated coverslips for 16 h, fixed, and stained for plectin, the integrin β4 subunit, and the LN5 γ2 chain. Note the accumulation of LN5-containing vesicles in the Tiam1−/− cells. Bars, 20 μm.

Mentions: Spreading of keratinocytes on glass requires the secretion and deposition of LN5 for initial adhesion. Keratinocytes bind to LN5 through the α3β1 integrin and increase their LN5 production and secretion during cell spreading. We first analyzed whether a lack of Tiam1 affected the intrinsic capacity of keratinocytes to produce LN5. In the absence of adhesive stimuli (suspended cells), an equal amount of protein (Fig. 8 A) and mRNA (Fig. 8 C) of LN5 (γ2 chain subunit) was found in WT and Tiam1−/− keratinocytes, indicating that the loss of Tiam1 expression did not affect the transcription and translation of LN5. We also analyzed the requirement of Tiam1 for the stimulation of LN5 production and secretion after initial adhesion. WT and Tiam1−/− keratinocytes were seeded on glass and the secreted matrix was scraped off the plates, size-separated by SDS-PAGE, and immunoblotted with a γ2-specific antibody. As shown in Fig. 8 (A and B), the amount of LN5 secreted by Tiam1−/− cells on glass for 30 and 180 min was strongly reduced as compared with WT cells. We also seeded these cells on an exogenous Col IV substrate to allow cell spreading. In WT cells, a consistent increase in the amount of LN5 mRNA (Fig. 8 C) was found after adhesion to Col IV. In contrast, no increase in LN5 mRNA was detected in Tiam1−/− keratinocytes (Fig. 8 C). Moreover, the amount of secreted LN5 on a Col IV matrix was strongly reduced in Tiam1−/− cells similarly as found when seeding these cells on glass (Fig. 8, A and B). To confirm that the increase in LN5 mRNA levels was because of Tiam1-mediated Rac activation, we analyzed Tiam1−/− keratinocytes that were reconstituted with Tiam1 and constitutive active V12Rac1 (Fig. 2). RT-PCR analysis revealed that both Tiam1 and V12Rac were able to rescue the increase in LN5 mRNA in Tiam1−/− cells to the level of WT cells (Fig. 8 C). Together these findings indicate that Tiam1-mediated Rac activation is required for the increase in levels of LN5 mRNA and protein upon adhesion of keratinocytes to an exogenous substrate. In addition, our data show that the LN5 secretion of Tiam1−/− keratinocytes upon adhesion to both glass and Col IV is impaired when compared with WT cells. Indeed, immunohistological analysis of WT keratinocytes with LN5 antibodies (γ2 chain subunit) revealed deposition of LN5 into regular archlike structures, partially colocalized with the integrin β4 subunit. In contrast, in Tiam1−/− cells most of the LN5 was retained in vesicles and only a small amount of secreted LN5 could be detected (Fig. 8 D). These data are consistent with the differences found in LN5 production and secretion using biochemical methods (Fig. 8, A–C). Together our findings suggest that Tiam1-Rac signaling regulates the increase in LN5 mRNA and protein levels, as well as the secretion of LN5. These processes are both essential for proper LN5 deposition and spreading of keratinocytes.


The Rac activator Tiam1 is required for (alpha)3(beta)1-mediated laminin-5 deposition, cell spreading, and cell migration.

Hamelers IH, Olivo C, Mertens AE, Pegtel DM, van der Kammen RA, Sonnenberg A, Collard JG - J. Cell Biol. (2005)

Production and secretion of LN5 by WT and Tiam1−/− keratinocytes. (A) Suspended WT and Tiam1−/− keratinocytes were seeded on a glass surface or on a Col IV–coated surface. After 30 or 180 min, attached cells were detached with 10 mM EDTA and secreted LN5 was detached from the surface with SDS–sample buffer. As a control, suspended cells were lysed. The samples were subjected to Western blotting and probed for LN5 (γ2 chain). (B) The amounts of secreted LN5 (γ2 chain) levels in the Western blot shown in A were quantified relative to levels in the 180-min WT sample (=100%) with Image J. (C) Suspended WT, Tiam1−/−, and Tiam1−/− keratinocytes stably expressing full-length Tiam1 or RacV12 were seeded on a Col IV–coated surface. A sample of the cells in suspension was used for mRNA isolation. After 45 min, adherent cells were detached from the surface and lysed for mRNA isolation. An RT-PCR reaction was performed to determine the amount of LN5 γ2 chain mRNA present in the samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control. The histogram represents the increase in LN5 mRNA levels in both WT and Tiam1−/− cells determined in two independent experiments. Error bars represent the SD. (D) Cells were seeded on Col IV–coated coverslips for 16 h, fixed, and stained for plectin, the integrin β4 subunit, and the LN5 γ2 chain. Note the accumulation of LN5-containing vesicles in the Tiam1−/− cells. Bars, 20 μm.
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fig8: Production and secretion of LN5 by WT and Tiam1−/− keratinocytes. (A) Suspended WT and Tiam1−/− keratinocytes were seeded on a glass surface or on a Col IV–coated surface. After 30 or 180 min, attached cells were detached with 10 mM EDTA and secreted LN5 was detached from the surface with SDS–sample buffer. As a control, suspended cells were lysed. The samples were subjected to Western blotting and probed for LN5 (γ2 chain). (B) The amounts of secreted LN5 (γ2 chain) levels in the Western blot shown in A were quantified relative to levels in the 180-min WT sample (=100%) with Image J. (C) Suspended WT, Tiam1−/−, and Tiam1−/− keratinocytes stably expressing full-length Tiam1 or RacV12 were seeded on a Col IV–coated surface. A sample of the cells in suspension was used for mRNA isolation. After 45 min, adherent cells were detached from the surface and lysed for mRNA isolation. An RT-PCR reaction was performed to determine the amount of LN5 γ2 chain mRNA present in the samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control. The histogram represents the increase in LN5 mRNA levels in both WT and Tiam1−/− cells determined in two independent experiments. Error bars represent the SD. (D) Cells were seeded on Col IV–coated coverslips for 16 h, fixed, and stained for plectin, the integrin β4 subunit, and the LN5 γ2 chain. Note the accumulation of LN5-containing vesicles in the Tiam1−/− cells. Bars, 20 μm.
Mentions: Spreading of keratinocytes on glass requires the secretion and deposition of LN5 for initial adhesion. Keratinocytes bind to LN5 through the α3β1 integrin and increase their LN5 production and secretion during cell spreading. We first analyzed whether a lack of Tiam1 affected the intrinsic capacity of keratinocytes to produce LN5. In the absence of adhesive stimuli (suspended cells), an equal amount of protein (Fig. 8 A) and mRNA (Fig. 8 C) of LN5 (γ2 chain subunit) was found in WT and Tiam1−/− keratinocytes, indicating that the loss of Tiam1 expression did not affect the transcription and translation of LN5. We also analyzed the requirement of Tiam1 for the stimulation of LN5 production and secretion after initial adhesion. WT and Tiam1−/− keratinocytes were seeded on glass and the secreted matrix was scraped off the plates, size-separated by SDS-PAGE, and immunoblotted with a γ2-specific antibody. As shown in Fig. 8 (A and B), the amount of LN5 secreted by Tiam1−/− cells on glass for 30 and 180 min was strongly reduced as compared with WT cells. We also seeded these cells on an exogenous Col IV substrate to allow cell spreading. In WT cells, a consistent increase in the amount of LN5 mRNA (Fig. 8 C) was found after adhesion to Col IV. In contrast, no increase in LN5 mRNA was detected in Tiam1−/− keratinocytes (Fig. 8 C). Moreover, the amount of secreted LN5 on a Col IV matrix was strongly reduced in Tiam1−/− cells similarly as found when seeding these cells on glass (Fig. 8, A and B). To confirm that the increase in LN5 mRNA levels was because of Tiam1-mediated Rac activation, we analyzed Tiam1−/− keratinocytes that were reconstituted with Tiam1 and constitutive active V12Rac1 (Fig. 2). RT-PCR analysis revealed that both Tiam1 and V12Rac were able to rescue the increase in LN5 mRNA in Tiam1−/− cells to the level of WT cells (Fig. 8 C). Together these findings indicate that Tiam1-mediated Rac activation is required for the increase in levels of LN5 mRNA and protein upon adhesion of keratinocytes to an exogenous substrate. In addition, our data show that the LN5 secretion of Tiam1−/− keratinocytes upon adhesion to both glass and Col IV is impaired when compared with WT cells. Indeed, immunohistological analysis of WT keratinocytes with LN5 antibodies (γ2 chain subunit) revealed deposition of LN5 into regular archlike structures, partially colocalized with the integrin β4 subunit. In contrast, in Tiam1−/− cells most of the LN5 was retained in vesicles and only a small amount of secreted LN5 could be detected (Fig. 8 D). These data are consistent with the differences found in LN5 production and secretion using biochemical methods (Fig. 8, A–C). Together our findings suggest that Tiam1-Rac signaling regulates the increase in LN5 mRNA and protein levels, as well as the secretion of LN5. These processes are both essential for proper LN5 deposition and spreading of keratinocytes.

Bottom Line: Both Tiam1 and V12Rac1 can rescue the defects of Tiam1-/- keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation.Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin.Our studies indicate that Tiam1 is a key molecule in alpha3beta1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
The Rho-like guanosine triphosphatase Rac1 regulates various signaling pathways, including integrin-mediated adhesion and migration of cells. However, the mechanisms by which integrins signal toward Rac are poorly understood. We show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis 1) is required for the integrin-mediated laminin (LN)-5 deposition, spreading, and migration of keratinocytes. In contrast to wild-type keratinocytes, Tiam1-deficient (Tiam1-/-) keratinocytes are unable to adhere to and spread on a glass substrate because they are unable to deposit their own LN5 substrate. Both Tiam1 and V12Rac1 can rescue the defects of Tiam1-/- keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation. Tiam1-/- cells are unable to activate Rac upon alpha3beta1-mediated adhesion to an exogenous LN5 substrate. Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin. Our studies indicate that Tiam1 is a key molecule in alpha3beta1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.

Show MeSH
Related in: MedlinePlus