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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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Phospho-Smad2 and Smad4 regulate LM α3 subunit gene transcription. (A) Differential phosphorylation of Smads. Subconfluent (Subcfl.) or confluent cultures either untreated (control) or treated with 5 ng/ml TGF-β1 for 6 h in the absence or presence of the TβR-I inhibitor SB431542 (+TGF-β1 or + TGF-β1+SB43, respectively) were analyzed by Western blotting for phospho-Smad2 (P-Smad2), total Smad2, P-Smad3, and total Smad3. (B) Phospho-Smad2 is localized to the nuclei after TGF-β1 treatment in confluent cells. Untreated (control) or TGF-β1–treated confluent cells (+TGF-β1) were stained with antibodies against P-Smad2 (red) and LM-332 (β3 subunit; green) and analyzed by confocal fluorescence microscopy. Nuclear staining with DAPI, blue. Bar, 10 μm. (C) Smad 4 is also localized to the nuclei in subconfluent and TGF-β1–treated confluent cells. Subconfluent MDCK cell cultures without added exogenous TGF-β1 and confluent cultures either untreated (control) or treated with of TGF-β1 in the presence or absence of SB431542 (SB) were stained with antibodies against Smad4 (green) and analyzed by confocal fluorescence microscopy. Nuclear staining with DAPI, blue. Bar, 10 μm. (D) Phosho-Smad2 and Smad4 form a complex dependent on TβR-I signaling. Confluent cultures treated with TGF-β1 to induce LM-332 expression in the absence or presence of the TβR-I inhibitor SB431542 (+TGF or +TGF+SB43, respectively) were extracted in RIPA buffer. Extracts were immunoprecipitated with an anti-Smad4 antibody (mouse) and Western blotted with anti-P-Smad2 antibody (rabbit). (E) P-Smad2 and Smad4 also form a complex in subconfluent cells. Extracts of subconfluent MDCK cell cultures without added exogenous TGF-β1 were immunoprecipitated with a control IgG (mock) or with an anti-Smad4 antibody (mouse), and Western blotted for P-Smad2 (rabbit). (F) DN Smad2 and Smad4, but not Smad3, impairs α3 subunit transcription in subconfluent cultures. Subconfluent MDCK cell cultures were transiently transfected with DN-Smad2, DN-Smad3, or DN-Smad4, and α3 subunit mRNA expression was analyzed by qRT-PCR after 24 h. **p < 0.05; ***p < 0.001 (G) Smad4 binds to an Smad binding element in the α3 subunit gene promoter. Subconfluent (6 h) or confluent (4 d) MDCK cell extracts were subjected to ChIP with anti-Smad4, RNA-polymerase II, or nonspecific antibodies (IgG). Enrichment of the SBE in the immunoprecipitated chromatin was determined by touchdown-PCR using primers specific for the α3 subunit promoter. Products were resolved by agarose gel electrophoresis (negative staining is shown) and compared with whole chromatin lysates (input).
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Figure 4: Phospho-Smad2 and Smad4 regulate LM α3 subunit gene transcription. (A) Differential phosphorylation of Smads. Subconfluent (Subcfl.) or confluent cultures either untreated (control) or treated with 5 ng/ml TGF-β1 for 6 h in the absence or presence of the TβR-I inhibitor SB431542 (+TGF-β1 or + TGF-β1+SB43, respectively) were analyzed by Western blotting for phospho-Smad2 (P-Smad2), total Smad2, P-Smad3, and total Smad3. (B) Phospho-Smad2 is localized to the nuclei after TGF-β1 treatment in confluent cells. Untreated (control) or TGF-β1–treated confluent cells (+TGF-β1) were stained with antibodies against P-Smad2 (red) and LM-332 (β3 subunit; green) and analyzed by confocal fluorescence microscopy. Nuclear staining with DAPI, blue. Bar, 10 μm. (C) Smad 4 is also localized to the nuclei in subconfluent and TGF-β1–treated confluent cells. Subconfluent MDCK cell cultures without added exogenous TGF-β1 and confluent cultures either untreated (control) or treated with of TGF-β1 in the presence or absence of SB431542 (SB) were stained with antibodies against Smad4 (green) and analyzed by confocal fluorescence microscopy. Nuclear staining with DAPI, blue. Bar, 10 μm. (D) Phosho-Smad2 and Smad4 form a complex dependent on TβR-I signaling. Confluent cultures treated with TGF-β1 to induce LM-332 expression in the absence or presence of the TβR-I inhibitor SB431542 (+TGF or +TGF+SB43, respectively) were extracted in RIPA buffer. Extracts were immunoprecipitated with an anti-Smad4 antibody (mouse) and Western blotted with anti-P-Smad2 antibody (rabbit). (E) P-Smad2 and Smad4 also form a complex in subconfluent cells. Extracts of subconfluent MDCK cell cultures without added exogenous TGF-β1 were immunoprecipitated with a control IgG (mock) or with an anti-Smad4 antibody (mouse), and Western blotted for P-Smad2 (rabbit). (F) DN Smad2 and Smad4, but not Smad3, impairs α3 subunit transcription in subconfluent cultures. Subconfluent MDCK cell cultures were transiently transfected with DN-Smad2, DN-Smad3, or DN-Smad4, and α3 subunit mRNA expression was analyzed by qRT-PCR after 24 h. **p < 0.05; ***p < 0.001 (G) Smad4 binds to an Smad binding element in the α3 subunit gene promoter. Subconfluent (6 h) or confluent (4 d) MDCK cell extracts were subjected to ChIP with anti-Smad4, RNA-polymerase II, or nonspecific antibodies (IgG). Enrichment of the SBE in the immunoprecipitated chromatin was determined by touchdown-PCR using primers specific for the α3 subunit promoter. Products were resolved by agarose gel electrophoresis (negative staining is shown) and compared with whole chromatin lysates (input).

Mentions: Ligated TGF-β1 type I and II receptors phosphorylate the so-called “receptor-activated” or R-Smads Smad 2 and -3. As shown in Figure 4A, phospho-Smad2 was detected in subconfluent MDCK cell cultures expressing LM-332 but not in confluent cultures. Stimulation of confluent cultures with exogenous activated TGF-β1–stimulated phosphorylation of Smad2, whereas inhibition of TβR-I with SB431542 blocked this phosphorylation. In contrast, Smad3 phosphorylation was low in subconfluent cultures, and baseline levels of Smad3 phosphorylation were only modestly affected by exogenous TGF-β1 and the inhibitor of TβR-I in confluent cultures (Figure 4A), suggesting that Smad2 but not Smad3 was involved in TGF-β1 signaling in MDCK cells under these conditions.


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)

Phospho-Smad2 and Smad4 regulate LM α3 subunit gene transcription. (A) Differential phosphorylation of Smads. Subconfluent (Subcfl.) or confluent cultures either untreated (control) or treated with 5 ng/ml TGF-β1 for 6 h in the absence or presence of the TβR-I inhibitor SB431542 (+TGF-β1 or + TGF-β1+SB43, respectively) were analyzed by Western blotting for phospho-Smad2 (P-Smad2), total Smad2, P-Smad3, and total Smad3. (B) Phospho-Smad2 is localized to the nuclei after TGF-β1 treatment in confluent cells. Untreated (control) or TGF-β1–treated confluent cells (+TGF-β1) were stained with antibodies against P-Smad2 (red) and LM-332 (β3 subunit; green) and analyzed by confocal fluorescence microscopy. Nuclear staining with DAPI, blue. Bar, 10 μm. (C) Smad 4 is also localized to the nuclei in subconfluent and TGF-β1–treated confluent cells. Subconfluent MDCK cell cultures without added exogenous TGF-β1 and confluent cultures either untreated (control) or treated with of TGF-β1 in the presence or absence of SB431542 (SB) were stained with antibodies against Smad4 (green) and analyzed by confocal fluorescence microscopy. Nuclear staining with DAPI, blue. Bar, 10 μm. (D) Phosho-Smad2 and Smad4 form a complex dependent on TβR-I signaling. Confluent cultures treated with TGF-β1 to induce LM-332 expression in the absence or presence of the TβR-I inhibitor SB431542 (+TGF or +TGF+SB43, respectively) were extracted in RIPA buffer. Extracts were immunoprecipitated with an anti-Smad4 antibody (mouse) and Western blotted with anti-P-Smad2 antibody (rabbit). (E) P-Smad2 and Smad4 also form a complex in subconfluent cells. Extracts of subconfluent MDCK cell cultures without added exogenous TGF-β1 were immunoprecipitated with a control IgG (mock) or with an anti-Smad4 antibody (mouse), and Western blotted for P-Smad2 (rabbit). (F) DN Smad2 and Smad4, but not Smad3, impairs α3 subunit transcription in subconfluent cultures. Subconfluent MDCK cell cultures were transiently transfected with DN-Smad2, DN-Smad3, or DN-Smad4, and α3 subunit mRNA expression was analyzed by qRT-PCR after 24 h. **p < 0.05; ***p < 0.001 (G) Smad4 binds to an Smad binding element in the α3 subunit gene promoter. Subconfluent (6 h) or confluent (4 d) MDCK cell extracts were subjected to ChIP with anti-Smad4, RNA-polymerase II, or nonspecific antibodies (IgG). Enrichment of the SBE in the immunoprecipitated chromatin was determined by touchdown-PCR using primers specific for the α3 subunit promoter. Products were resolved by agarose gel electrophoresis (negative staining is shown) and compared with whole chromatin lysates (input).
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Figure 4: Phospho-Smad2 and Smad4 regulate LM α3 subunit gene transcription. (A) Differential phosphorylation of Smads. Subconfluent (Subcfl.) or confluent cultures either untreated (control) or treated with 5 ng/ml TGF-β1 for 6 h in the absence or presence of the TβR-I inhibitor SB431542 (+TGF-β1 or + TGF-β1+SB43, respectively) were analyzed by Western blotting for phospho-Smad2 (P-Smad2), total Smad2, P-Smad3, and total Smad3. (B) Phospho-Smad2 is localized to the nuclei after TGF-β1 treatment in confluent cells. Untreated (control) or TGF-β1–treated confluent cells (+TGF-β1) were stained with antibodies against P-Smad2 (red) and LM-332 (β3 subunit; green) and analyzed by confocal fluorescence microscopy. Nuclear staining with DAPI, blue. Bar, 10 μm. (C) Smad 4 is also localized to the nuclei in subconfluent and TGF-β1–treated confluent cells. Subconfluent MDCK cell cultures without added exogenous TGF-β1 and confluent cultures either untreated (control) or treated with of TGF-β1 in the presence or absence of SB431542 (SB) were stained with antibodies against Smad4 (green) and analyzed by confocal fluorescence microscopy. Nuclear staining with DAPI, blue. Bar, 10 μm. (D) Phosho-Smad2 and Smad4 form a complex dependent on TβR-I signaling. Confluent cultures treated with TGF-β1 to induce LM-332 expression in the absence or presence of the TβR-I inhibitor SB431542 (+TGF or +TGF+SB43, respectively) were extracted in RIPA buffer. Extracts were immunoprecipitated with an anti-Smad4 antibody (mouse) and Western blotted with anti-P-Smad2 antibody (rabbit). (E) P-Smad2 and Smad4 also form a complex in subconfluent cells. Extracts of subconfluent MDCK cell cultures without added exogenous TGF-β1 were immunoprecipitated with a control IgG (mock) or with an anti-Smad4 antibody (mouse), and Western blotted for P-Smad2 (rabbit). (F) DN Smad2 and Smad4, but not Smad3, impairs α3 subunit transcription in subconfluent cultures. Subconfluent MDCK cell cultures were transiently transfected with DN-Smad2, DN-Smad3, or DN-Smad4, and α3 subunit mRNA expression was analyzed by qRT-PCR after 24 h. **p < 0.05; ***p < 0.001 (G) Smad4 binds to an Smad binding element in the α3 subunit gene promoter. Subconfluent (6 h) or confluent (4 d) MDCK cell extracts were subjected to ChIP with anti-Smad4, RNA-polymerase II, or nonspecific antibodies (IgG). Enrichment of the SBE in the immunoprecipitated chromatin was determined by touchdown-PCR using primers specific for the α3 subunit promoter. Products were resolved by agarose gel electrophoresis (negative staining is shown) and compared with whole chromatin lysates (input).
Mentions: Ligated TGF-β1 type I and II receptors phosphorylate the so-called “receptor-activated” or R-Smads Smad 2 and -3. As shown in Figure 4A, phospho-Smad2 was detected in subconfluent MDCK cell cultures expressing LM-332 but not in confluent cultures. Stimulation of confluent cultures with exogenous activated TGF-β1–stimulated phosphorylation of Smad2, whereas inhibition of TβR-I with SB431542 blocked this phosphorylation. In contrast, Smad3 phosphorylation was low in subconfluent cultures, and baseline levels of Smad3 phosphorylation were only modestly affected by exogenous TGF-β1 and the inhibitor of TβR-I in confluent cultures (Figure 4A), suggesting that Smad2 but not Smad3 was involved in TGF-β1 signaling in MDCK cells under these conditions.

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