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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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Laminin-332 (LM-332) expression is regulated as a function of confluence. (A) LM-332 deposition only occurs in subconfluent cells. Subconfluent (day 1) or confluent (day 4) MDCK cells cultured on 0.4-μm Transwell supports were immunostained for LM-332 with an anti-β3 subunit mAb (green). Confocal sections corresponding to the basal plane (deposited extracellular matrix [ECM]) are shown. Bar, 10 μm. (B) Significant amounts of LM-332 are deposited into the substratum only in subconfluent (day 1) cultures. Cells plated on Transwell supports were removed by treatment with 20 mM NH4OH at the indicated time points (days 1–7). Deposited ECM proteins were extracted, resolved by SDS-polyacrylamide gel electrophoresis and Western-blotted for LM-332 with an anti-β3 mAb. (C) Laminin α3 and γ2 subunit expression is transcriptionally regulated as a function of cell confluence. RNA from Transwell cultures was isolated at different time points (days 1–4) and analyzed by qRT-PCR using canine-specific primers. Inset, qRT-PCR for the α5 subunit of LM-511 (LM-α5). The histograms represent the average abundance of mRNAs from three independent experiments expressed in picograms ± SD. **p < 0.01 or *p < 0.05 relative to day 1 (d1) levels.
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Figure 1: Laminin-332 (LM-332) expression is regulated as a function of confluence. (A) LM-332 deposition only occurs in subconfluent cells. Subconfluent (day 1) or confluent (day 4) MDCK cells cultured on 0.4-μm Transwell supports were immunostained for LM-332 with an anti-β3 subunit mAb (green). Confocal sections corresponding to the basal plane (deposited extracellular matrix [ECM]) are shown. Bar, 10 μm. (B) Significant amounts of LM-332 are deposited into the substratum only in subconfluent (day 1) cultures. Cells plated on Transwell supports were removed by treatment with 20 mM NH4OH at the indicated time points (days 1–7). Deposited ECM proteins were extracted, resolved by SDS-polyacrylamide gel electrophoresis and Western-blotted for LM-332 with an anti-β3 mAb. (C) Laminin α3 and γ2 subunit expression is transcriptionally regulated as a function of cell confluence. RNA from Transwell cultures was isolated at different time points (days 1–4) and analyzed by qRT-PCR using canine-specific primers. Inset, qRT-PCR for the α5 subunit of LM-511 (LM-α5). The histograms represent the average abundance of mRNAs from three independent experiments expressed in picograms ± SD. **p < 0.01 or *p < 0.05 relative to day 1 (d1) levels.

Mentions: As shown in Figure 1, LM-332 was visible in a basal optical section by confocal immunofluorescence microscopy only in a subconfluent (day 1) culture but not in a confluent (day 4) culture (Figure 1A). Similarly, LM-332 (as detected by Western blotting with an antibody against the β3 subunit) was deposited onto the permeable support in significant amounts at day 1 of culture but was present in diminishing amounts at day 4 and, to an even lesser extent, at day 7, suggesting that not only was secretion and deposition of LM-332 reduced as confluence was achieved but also that any residual LM-332 was removed (Figure 1B).


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)

Laminin-332 (LM-332) expression is regulated as a function of confluence. (A) LM-332 deposition only occurs in subconfluent cells. Subconfluent (day 1) or confluent (day 4) MDCK cells cultured on 0.4-μm Transwell supports were immunostained for LM-332 with an anti-β3 subunit mAb (green). Confocal sections corresponding to the basal plane (deposited extracellular matrix [ECM]) are shown. Bar, 10 μm. (B) Significant amounts of LM-332 are deposited into the substratum only in subconfluent (day 1) cultures. Cells plated on Transwell supports were removed by treatment with 20 mM NH4OH at the indicated time points (days 1–7). Deposited ECM proteins were extracted, resolved by SDS-polyacrylamide gel electrophoresis and Western-blotted for LM-332 with an anti-β3 mAb. (C) Laminin α3 and γ2 subunit expression is transcriptionally regulated as a function of cell confluence. RNA from Transwell cultures was isolated at different time points (days 1–4) and analyzed by qRT-PCR using canine-specific primers. Inset, qRT-PCR for the α5 subunit of LM-511 (LM-α5). The histograms represent the average abundance of mRNAs from three independent experiments expressed in picograms ± SD. **p < 0.01 or *p < 0.05 relative to day 1 (d1) levels.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Laminin-332 (LM-332) expression is regulated as a function of confluence. (A) LM-332 deposition only occurs in subconfluent cells. Subconfluent (day 1) or confluent (day 4) MDCK cells cultured on 0.4-μm Transwell supports were immunostained for LM-332 with an anti-β3 subunit mAb (green). Confocal sections corresponding to the basal plane (deposited extracellular matrix [ECM]) are shown. Bar, 10 μm. (B) Significant amounts of LM-332 are deposited into the substratum only in subconfluent (day 1) cultures. Cells plated on Transwell supports were removed by treatment with 20 mM NH4OH at the indicated time points (days 1–7). Deposited ECM proteins were extracted, resolved by SDS-polyacrylamide gel electrophoresis and Western-blotted for LM-332 with an anti-β3 mAb. (C) Laminin α3 and γ2 subunit expression is transcriptionally regulated as a function of cell confluence. RNA from Transwell cultures was isolated at different time points (days 1–4) and analyzed by qRT-PCR using canine-specific primers. Inset, qRT-PCR for the α5 subunit of LM-511 (LM-α5). The histograms represent the average abundance of mRNAs from three independent experiments expressed in picograms ± SD. **p < 0.01 or *p < 0.05 relative to day 1 (d1) levels.
Mentions: As shown in Figure 1, LM-332 was visible in a basal optical section by confocal immunofluorescence microscopy only in a subconfluent (day 1) culture but not in a confluent (day 4) culture (Figure 1A). Similarly, LM-332 (as detected by Western blotting with an antibody against the β3 subunit) was deposited onto the permeable support in significant amounts at day 1 of culture but was present in diminishing amounts at day 4 and, to an even lesser extent, at day 7, suggesting that not only was secretion and deposition of LM-332 reduced as confluence was achieved but also that any residual LM-332 was removed (Figure 1B).

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