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Autocrine transforming growth factor-{beta}1 activation mediated by integrin {alpha}V{beta}3 regulates transcriptional expression of laminin-332 in Madin-Darby canine kidney epithelial cells.

Moyano JV, Greciano PG, Buschmann MM, Koch M, Matlin KS - Mol. Biol. Cell (2010)

Bottom Line: Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-β1 secretion and activation mediated by integrin αVβ3 because neutralizing antibodies block LM-332 production in subconfluent cells.In confluent cells, latent TGF-β1 is secreted apically, whereas TβR-I and integrin αVβ3 are localized basolaterally.Disruption of the epithelial barrier by mechanical injury activates TGF-β1, leading to LM-332 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Committee on Cell Physiology, and Committee on Molecular Pathogenesis and Molecular Medicine, The University of Chicago, Chicago, IL 60637, USA. jvmoyano@uchicago.edu

ABSTRACT
Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed. Addition of active transforming growth factor (TGF)-β1 to confluent MDCK monolayers is sufficient to induce transcription of the LM α3 gene and LM-332 protein expression via the TGF-β type I receptor (TβR-I) and the Smad2-Smad4 complex. Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-β1 secretion and activation mediated by integrin αVβ3 because neutralizing antibodies block LM-332 production in subconfluent cells. In confluent cells, latent TGF-β1 is secreted apically, whereas TβR-I and integrin αVβ3 are localized basolaterally. Disruption of the epithelial barrier by mechanical injury activates TGF-β1, leading to LM-332 expression. Together, our data suggest a novel mechanism for triggering the production of LM-332 after epithelial injury.

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Exogenous active TGF-β1 is sufficient to induce LM-332 expression in confluent cells. (A) TGF-β1 induces transcription of the laminin α3 subunit gene. Confluent MDCK cells untreated (control) or treated with 5 ng/ml active TGF-β1 for 6 h in serum-free medium (+TGF-β1) were collected and analyzed for laminin α3 subunit mRNA by q-RT-PCR. **p = 0.0011 (B) TGF-β1 induces LM-332 protein synthesis. i, untreated control (control) or TGF-β1-treated (+TGF-β1) confluent cultures were metabolically labeled with [35S]Met/Cys for 20 min and extracted with RIPA buffer. The extracts were immunoprecipitated with a polyclonal antibody against LM-332, and the immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and detected by autoradiography. The graph represents the intensities of the α3 bands in this experiment. ii, equal amounts of total protein from RIPA lysates were analyzed by Western blot for LM-332 using a mAb against the β3 subunit with immunoblotting for tubulin as loading control. (C) Confocal immunofluorescence localization of LM-332 after TGF-β1 treatment. Subconfluent and confluent MDCK cell cultures without added exogenous TGF-β1, and TGF-β1-treated confluent MDCK cells were immunostained with a polyclonal antibody against LM-332 (red) and optical sections of a mid/basal plane visualized. Nuclei were stained with DAPI (blue). Bar, 10 μm. (D) Transepithelial resistance of MDCK cells is not affected by TGF-β1 treatment. Transepithelial electrical resistance of confluent MDCK cultures grown on Transwell supports in either the absence or presence of exogenous active TGF-β1 was measured with a Millicell-ERS device. The experiment was repeated four times, and the average is shown. n.s., not statistically significant, p = 0.1743.
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Figure 2: Exogenous active TGF-β1 is sufficient to induce LM-332 expression in confluent cells. (A) TGF-β1 induces transcription of the laminin α3 subunit gene. Confluent MDCK cells untreated (control) or treated with 5 ng/ml active TGF-β1 for 6 h in serum-free medium (+TGF-β1) were collected and analyzed for laminin α3 subunit mRNA by q-RT-PCR. **p = 0.0011 (B) TGF-β1 induces LM-332 protein synthesis. i, untreated control (control) or TGF-β1-treated (+TGF-β1) confluent cultures were metabolically labeled with [35S]Met/Cys for 20 min and extracted with RIPA buffer. The extracts were immunoprecipitated with a polyclonal antibody against LM-332, and the immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and detected by autoradiography. The graph represents the intensities of the α3 bands in this experiment. ii, equal amounts of total protein from RIPA lysates were analyzed by Western blot for LM-332 using a mAb against the β3 subunit with immunoblotting for tubulin as loading control. (C) Confocal immunofluorescence localization of LM-332 after TGF-β1 treatment. Subconfluent and confluent MDCK cell cultures without added exogenous TGF-β1, and TGF-β1-treated confluent MDCK cells were immunostained with a polyclonal antibody against LM-332 (red) and optical sections of a mid/basal plane visualized. Nuclei were stained with DAPI (blue). Bar, 10 μm. (D) Transepithelial resistance of MDCK cells is not affected by TGF-β1 treatment. Transepithelial electrical resistance of confluent MDCK cultures grown on Transwell supports in either the absence or presence of exogenous active TGF-β1 was measured with a Millicell-ERS device. The experiment was repeated four times, and the average is shown. n.s., not statistically significant, p = 0.1743.

Mentions: To test the involvement of TGF-β1 in regulation of LM-332 production in MDCK cells, exogenous activated TGF-β1 was added both apically and basolaterally to confluent (day 4) cultures of MDCK cells that normally do not express LM-332 (Figure 1). As illustrated in Figure 2A, addition of TGF-β1 for 6 h stimulated transcription of mRNA for the α3 subunit. In addition, increased synthesis of LM-332 was also evident in similarly treated cultures when analyzed either by metabolic-labeling and immunoprecipitation or Western blotting (Figure 2B). These findings were further supported by detection of LM-332 in treated cells by immunofluorescence (Figure 2C). Treatment with exogenous TGF-β1 did not compromise the epithelial barrier, as determined by measurement of transepithelial resistance (Figure 2D). Thus, under conditions in which MDCK cells do not normally express LM-332, TGF-β1 treatment induces LM-332 mRNA transcription and protein synthesis, suggesting a possible physiological role for TGF-β1 in the regulation of LM-332 expression.


Autocrine transforming growth factor-{beta}1 activation mediated by integrin {alpha}V{beta}3 regulates transcriptional expression of laminin-332 in Madin-Darby canine kidney epithelial cells.

Moyano JV, Greciano PG, Buschmann MM, Koch M, Matlin KS - Mol. Biol. Cell (2010)

Exogenous active TGF-β1 is sufficient to induce LM-332 expression in confluent cells. (A) TGF-β1 induces transcription of the laminin α3 subunit gene. Confluent MDCK cells untreated (control) or treated with 5 ng/ml active TGF-β1 for 6 h in serum-free medium (+TGF-β1) were collected and analyzed for laminin α3 subunit mRNA by q-RT-PCR. **p = 0.0011 (B) TGF-β1 induces LM-332 protein synthesis. i, untreated control (control) or TGF-β1-treated (+TGF-β1) confluent cultures were metabolically labeled with [35S]Met/Cys for 20 min and extracted with RIPA buffer. The extracts were immunoprecipitated with a polyclonal antibody against LM-332, and the immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and detected by autoradiography. The graph represents the intensities of the α3 bands in this experiment. ii, equal amounts of total protein from RIPA lysates were analyzed by Western blot for LM-332 using a mAb against the β3 subunit with immunoblotting for tubulin as loading control. (C) Confocal immunofluorescence localization of LM-332 after TGF-β1 treatment. Subconfluent and confluent MDCK cell cultures without added exogenous TGF-β1, and TGF-β1-treated confluent MDCK cells were immunostained with a polyclonal antibody against LM-332 (red) and optical sections of a mid/basal plane visualized. Nuclei were stained with DAPI (blue). Bar, 10 μm. (D) Transepithelial resistance of MDCK cells is not affected by TGF-β1 treatment. Transepithelial electrical resistance of confluent MDCK cultures grown on Transwell supports in either the absence or presence of exogenous active TGF-β1 was measured with a Millicell-ERS device. The experiment was repeated four times, and the average is shown. n.s., not statistically significant, p = 0.1743.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Exogenous active TGF-β1 is sufficient to induce LM-332 expression in confluent cells. (A) TGF-β1 induces transcription of the laminin α3 subunit gene. Confluent MDCK cells untreated (control) or treated with 5 ng/ml active TGF-β1 for 6 h in serum-free medium (+TGF-β1) were collected and analyzed for laminin α3 subunit mRNA by q-RT-PCR. **p = 0.0011 (B) TGF-β1 induces LM-332 protein synthesis. i, untreated control (control) or TGF-β1-treated (+TGF-β1) confluent cultures were metabolically labeled with [35S]Met/Cys for 20 min and extracted with RIPA buffer. The extracts were immunoprecipitated with a polyclonal antibody against LM-332, and the immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and detected by autoradiography. The graph represents the intensities of the α3 bands in this experiment. ii, equal amounts of total protein from RIPA lysates were analyzed by Western blot for LM-332 using a mAb against the β3 subunit with immunoblotting for tubulin as loading control. (C) Confocal immunofluorescence localization of LM-332 after TGF-β1 treatment. Subconfluent and confluent MDCK cell cultures without added exogenous TGF-β1, and TGF-β1-treated confluent MDCK cells were immunostained with a polyclonal antibody against LM-332 (red) and optical sections of a mid/basal plane visualized. Nuclei were stained with DAPI (blue). Bar, 10 μm. (D) Transepithelial resistance of MDCK cells is not affected by TGF-β1 treatment. Transepithelial electrical resistance of confluent MDCK cultures grown on Transwell supports in either the absence or presence of exogenous active TGF-β1 was measured with a Millicell-ERS device. The experiment was repeated four times, and the average is shown. n.s., not statistically significant, p = 0.1743.
Mentions: To test the involvement of TGF-β1 in regulation of LM-332 production in MDCK cells, exogenous activated TGF-β1 was added both apically and basolaterally to confluent (day 4) cultures of MDCK cells that normally do not express LM-332 (Figure 1). As illustrated in Figure 2A, addition of TGF-β1 for 6 h stimulated transcription of mRNA for the α3 subunit. In addition, increased synthesis of LM-332 was also evident in similarly treated cultures when analyzed either by metabolic-labeling and immunoprecipitation or Western blotting (Figure 2B). These findings were further supported by detection of LM-332 in treated cells by immunofluorescence (Figure 2C). Treatment with exogenous TGF-β1 did not compromise the epithelial barrier, as determined by measurement of transepithelial resistance (Figure 2D). Thus, under conditions in which MDCK cells do not normally express LM-332, TGF-β1 treatment induces LM-332 mRNA transcription and protein synthesis, suggesting a possible physiological role for TGF-β1 in the regulation of LM-332 expression.

Bottom Line: Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-β1 secretion and activation mediated by integrin αVβ3 because neutralizing antibodies block LM-332 production in subconfluent cells.In confluent cells, latent TGF-β1 is secreted apically, whereas TβR-I and integrin αVβ3 are localized basolaterally.Disruption of the epithelial barrier by mechanical injury activates TGF-β1, leading to LM-332 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Committee on Cell Physiology, and Committee on Molecular Pathogenesis and Molecular Medicine, The University of Chicago, Chicago, IL 60637, USA. jvmoyano@uchicago.edu

ABSTRACT
Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed. Addition of active transforming growth factor (TGF)-β1 to confluent MDCK monolayers is sufficient to induce transcription of the LM α3 gene and LM-332 protein expression via the TGF-β type I receptor (TβR-I) and the Smad2-Smad4 complex. Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-β1 secretion and activation mediated by integrin αVβ3 because neutralizing antibodies block LM-332 production in subconfluent cells. In confluent cells, latent TGF-β1 is secreted apically, whereas TβR-I and integrin αVβ3 are localized basolaterally. Disruption of the epithelial barrier by mechanical injury activates TGF-β1, leading to LM-332 expression. Together, our data suggest a novel mechanism for triggering the production of LM-332 after epithelial injury.

Show MeSH
Related in: MedlinePlus