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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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Kinase activity of the TGF-β receptor type I (TβR-I) is required for LM-332 expression. (A) TβR-I is localized basolaterally in polarized MDCK cells. Confluent cultures of MDCK cells grown on permeable supports were biotinylated either apically (AP) or basolaterally (BL). Biotinylated proteins were captured on streptavidin-conjugated beads and immunoblotted with anti-TβR-I antibodies. The unbound protein fraction was used for immunoblotting with an mAb against tubulin as loading control. These experiments were repeated twice with similar results. (B) Inhibition of TβR-I signaling abolishes laminin α3 subunit mRNA expression. Confluent MDCK cells were pretreated with DMSO (control) or with 5 μM TβR-I kinase activity inhibitor SB431542 (+SB43) for 30 min. Then, cells were either incubated with or without exogenous TGF-β1 for 6 h and analyzed for laminin α3 subunit mRNA expression by qRT-PCR. n.s., not statistically significant; ***p < 0.001; **p < 0.01 (C) LM-332 protein expression is also diminished after blocking TβR-I signaling in TGF-β1–treated confluent MDCK cells. Confluent cultures of MDCK cells untreated or pretreated with SB431542 were then incubated with or without exogenous TGF-β1 for 6 h, and LM-332 expression was detected by immunofluorescence. Control/confluent, no TGF-β1 or SB431542; control/confluent + TGF-β1, treated with TGF-β1 but no SB431542; +SB431452/confluent+TGF-β1, treated with both TGF-β1 and SB431452. Bar, 10 μm. (D) Production of LM-332 was examined by Western blotting of MDCK cell extracts using an anti-β3 subunit monoclonal from cultures treated as described in C. SB43, treated with SB431452. (E) LM-332 expression is also dependent on TβR-I signaling in subconfluent cells. Subconfluent MDCK cell cultures without addition of exogenous TGF-β1 were incubated with or without SB431452 (SB43) for 18 h, and extracts were Western blotted for the β3 subunit of LM-332.
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Figure 3: Kinase activity of the TGF-β receptor type I (TβR-I) is required for LM-332 expression. (A) TβR-I is localized basolaterally in polarized MDCK cells. Confluent cultures of MDCK cells grown on permeable supports were biotinylated either apically (AP) or basolaterally (BL). Biotinylated proteins were captured on streptavidin-conjugated beads and immunoblotted with anti-TβR-I antibodies. The unbound protein fraction was used for immunoblotting with an mAb against tubulin as loading control. These experiments were repeated twice with similar results. (B) Inhibition of TβR-I signaling abolishes laminin α3 subunit mRNA expression. Confluent MDCK cells were pretreated with DMSO (control) or with 5 μM TβR-I kinase activity inhibitor SB431542 (+SB43) for 30 min. Then, cells were either incubated with or without exogenous TGF-β1 for 6 h and analyzed for laminin α3 subunit mRNA expression by qRT-PCR. n.s., not statistically significant; ***p < 0.001; **p < 0.01 (C) LM-332 protein expression is also diminished after blocking TβR-I signaling in TGF-β1–treated confluent MDCK cells. Confluent cultures of MDCK cells untreated or pretreated with SB431542 were then incubated with or without exogenous TGF-β1 for 6 h, and LM-332 expression was detected by immunofluorescence. Control/confluent, no TGF-β1 or SB431542; control/confluent + TGF-β1, treated with TGF-β1 but no SB431542; +SB431452/confluent+TGF-β1, treated with both TGF-β1 and SB431452. Bar, 10 μm. (D) Production of LM-332 was examined by Western blotting of MDCK cell extracts using an anti-β3 subunit monoclonal from cultures treated as described in C. SB43, treated with SB431452. (E) LM-332 expression is also dependent on TβR-I signaling in subconfluent cells. Subconfluent MDCK cell cultures without addition of exogenous TGF-β1 were incubated with or without SB431452 (SB43) for 18 h, and extracts were Western blotted for the β3 subunit of LM-332.

Mentions: TGF-β normally signals through type I and II receptors that are believed to form a heterotetramer on the cell surface when ligated (Wrana et al., 2008). In MDCK cells, the TβR-I receptor was expressed on the basolateral surface of confluent cultures, as determined by domain-selective biotinylation and Western blotting (Figure 3A), confirming previous results (Murphy et al., 2004). To ascertain whether this receptor is used by MDCK cells in the expression of LM-332, cells were preincubated with SB431542, a selective inhibitor of activin receptor-like kinase (ALK) superfamily members, including TβR-I (Inman et al., 2002), for 30 min before addition of exogenous activated TGF-β1 for 6 h to both the apical and basolateral chambers of a confluent Transwell culture of MDCK cells. As shown in Figure 3B, transcription of the laminin α3 gene, as determined by qRT-PCR, and synthesis of LM-332, as determined by immunofluorescence and Western blotting, was nearly abolished in samples treated with exogenous TGF-β1 and the TβR-I receptor inhibitor relative to control cultures treated only with TGF-β1 and vehicle (Figure 3, B–D). Furthermore, when subconfluent cultures of MDCK cells, which synthesize LM-332 in the absence of exogenous TGF-β1 stimulation, were treated with the inhibitor under the same conditions, LM-332 synthesis was significantly diminished (Figure 3E). Overall, these results strongly suggest that the TGF-β1 receptor TβR-I is mediating signals leading to the expression of LM-332 in MDCK cells.


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)

Kinase activity of the TGF-β receptor type I (TβR-I) is required for LM-332 expression. (A) TβR-I is localized basolaterally in polarized MDCK cells. Confluent cultures of MDCK cells grown on permeable supports were biotinylated either apically (AP) or basolaterally (BL). Biotinylated proteins were captured on streptavidin-conjugated beads and immunoblotted with anti-TβR-I antibodies. The unbound protein fraction was used for immunoblotting with an mAb against tubulin as loading control. These experiments were repeated twice with similar results. (B) Inhibition of TβR-I signaling abolishes laminin α3 subunit mRNA expression. Confluent MDCK cells were pretreated with DMSO (control) or with 5 μM TβR-I kinase activity inhibitor SB431542 (+SB43) for 30 min. Then, cells were either incubated with or without exogenous TGF-β1 for 6 h and analyzed for laminin α3 subunit mRNA expression by qRT-PCR. n.s., not statistically significant; ***p < 0.001; **p < 0.01 (C) LM-332 protein expression is also diminished after blocking TβR-I signaling in TGF-β1–treated confluent MDCK cells. Confluent cultures of MDCK cells untreated or pretreated with SB431542 were then incubated with or without exogenous TGF-β1 for 6 h, and LM-332 expression was detected by immunofluorescence. Control/confluent, no TGF-β1 or SB431542; control/confluent + TGF-β1, treated with TGF-β1 but no SB431542; +SB431452/confluent+TGF-β1, treated with both TGF-β1 and SB431452. Bar, 10 μm. (D) Production of LM-332 was examined by Western blotting of MDCK cell extracts using an anti-β3 subunit monoclonal from cultures treated as described in C. SB43, treated with SB431452. (E) LM-332 expression is also dependent on TβR-I signaling in subconfluent cells. Subconfluent MDCK cell cultures without addition of exogenous TGF-β1 were incubated with or without SB431452 (SB43) for 18 h, and extracts were Western blotted for the β3 subunit of LM-332.
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Figure 3: Kinase activity of the TGF-β receptor type I (TβR-I) is required for LM-332 expression. (A) TβR-I is localized basolaterally in polarized MDCK cells. Confluent cultures of MDCK cells grown on permeable supports were biotinylated either apically (AP) or basolaterally (BL). Biotinylated proteins were captured on streptavidin-conjugated beads and immunoblotted with anti-TβR-I antibodies. The unbound protein fraction was used for immunoblotting with an mAb against tubulin as loading control. These experiments were repeated twice with similar results. (B) Inhibition of TβR-I signaling abolishes laminin α3 subunit mRNA expression. Confluent MDCK cells were pretreated with DMSO (control) or with 5 μM TβR-I kinase activity inhibitor SB431542 (+SB43) for 30 min. Then, cells were either incubated with or without exogenous TGF-β1 for 6 h and analyzed for laminin α3 subunit mRNA expression by qRT-PCR. n.s., not statistically significant; ***p < 0.001; **p < 0.01 (C) LM-332 protein expression is also diminished after blocking TβR-I signaling in TGF-β1–treated confluent MDCK cells. Confluent cultures of MDCK cells untreated or pretreated with SB431542 were then incubated with or without exogenous TGF-β1 for 6 h, and LM-332 expression was detected by immunofluorescence. Control/confluent, no TGF-β1 or SB431542; control/confluent + TGF-β1, treated with TGF-β1 but no SB431542; +SB431452/confluent+TGF-β1, treated with both TGF-β1 and SB431452. Bar, 10 μm. (D) Production of LM-332 was examined by Western blotting of MDCK cell extracts using an anti-β3 subunit monoclonal from cultures treated as described in C. SB43, treated with SB431452. (E) LM-332 expression is also dependent on TβR-I signaling in subconfluent cells. Subconfluent MDCK cell cultures without addition of exogenous TGF-β1 were incubated with or without SB431452 (SB43) for 18 h, and extracts were Western blotted for the β3 subunit of LM-332.
Mentions: TGF-β normally signals through type I and II receptors that are believed to form a heterotetramer on the cell surface when ligated (Wrana et al., 2008). In MDCK cells, the TβR-I receptor was expressed on the basolateral surface of confluent cultures, as determined by domain-selective biotinylation and Western blotting (Figure 3A), confirming previous results (Murphy et al., 2004). To ascertain whether this receptor is used by MDCK cells in the expression of LM-332, cells were preincubated with SB431542, a selective inhibitor of activin receptor-like kinase (ALK) superfamily members, including TβR-I (Inman et al., 2002), for 30 min before addition of exogenous activated TGF-β1 for 6 h to both the apical and basolateral chambers of a confluent Transwell culture of MDCK cells. As shown in Figure 3B, transcription of the laminin α3 gene, as determined by qRT-PCR, and synthesis of LM-332, as determined by immunofluorescence and Western blotting, was nearly abolished in samples treated with exogenous TGF-β1 and the TβR-I receptor inhibitor relative to control cultures treated only with TGF-β1 and vehicle (Figure 3, B–D). Furthermore, when subconfluent cultures of MDCK cells, which synthesize LM-332 in the absence of exogenous TGF-β1 stimulation, were treated with the inhibitor under the same conditions, LM-332 synthesis was significantly diminished (Figure 3E). Overall, these results strongly suggest that the TGF-β1 receptor TβR-I is mediating signals leading to the expression of LM-332 in MDCK cells.

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