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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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Wounded confluent MDCK monolayers express LM-332. (A) Wounded epithelial monolayers activate TGF-β1. Conditioned media from a confluent MDCK cell culture (control) and a culture wounded by scraping with a pipette tip were analyzed by sandwich ELISA for total and active TGF-β1. **p < 0.01 (B) LM-332 expression is localized to the cells close to the wound edge. A confluent MDCK cell monolayer (control) and a culture injured by scraping (Wound) were analyzed after 6 h for LM-332 expression (green; left) or P-Smad2 (green/arrows; right). F-actin, red; nuclei, blue. Bar, 25 μm. (C) LM-332 synthesis is up-regulated in wounded cultures. Confluent MDCK cell monolayers (Ctrl) or wounded cultures (Wnd) were metabolically labeled with [35S]Met/Cys for 20 min 6 h after wounding, and synthesized LM-332 was analyzed by immunoprecipitation. Autoradiography revealed increased levels of α3, β3, and γ2 subunit synthesis in wounded monolayers compared with the unwounded control. The numbers between parentheses indicate fold-increase in band intensity compared with the control. Note that α3 and γ2 subunits show a similar fold-increase, consistent with coregulation of the α3 and γ2 mRNAs. (D) Blocking αVβ3 integrin with LM609 or RGD prevents TGF-β1 activation and LM-α3 mRNA expression. Confluent MDCK cell monolayers were treated with either anti-αVβ3 function blocking antibody (LM609), a nonspecific antibody (IgG), RGD or RGE peptides in ExCell. After 6 h, the conditioned media were analyzed for TGF-β1 activation (gray bars), and the cells were analyzed for LM-α3 mRNA expression by qRT-PCR (black bars). n.s., not statistically significant compared with “wound”; *p < 0.05; **p < 0.01 (E) Wounded MDCK monolayers treated as described in D were analyzed by immunofluorescence for LM-332 with a polyclonal antibody (green). F-actin, red; nuclei, blue. Bar, 25 μm.
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Figure 8: Wounded confluent MDCK monolayers express LM-332. (A) Wounded epithelial monolayers activate TGF-β1. Conditioned media from a confluent MDCK cell culture (control) and a culture wounded by scraping with a pipette tip were analyzed by sandwich ELISA for total and active TGF-β1. **p < 0.01 (B) LM-332 expression is localized to the cells close to the wound edge. A confluent MDCK cell monolayer (control) and a culture injured by scraping (Wound) were analyzed after 6 h for LM-332 expression (green; left) or P-Smad2 (green/arrows; right). F-actin, red; nuclei, blue. Bar, 25 μm. (C) LM-332 synthesis is up-regulated in wounded cultures. Confluent MDCK cell monolayers (Ctrl) or wounded cultures (Wnd) were metabolically labeled with [35S]Met/Cys for 20 min 6 h after wounding, and synthesized LM-332 was analyzed by immunoprecipitation. Autoradiography revealed increased levels of α3, β3, and γ2 subunit synthesis in wounded monolayers compared with the unwounded control. The numbers between parentheses indicate fold-increase in band intensity compared with the control. Note that α3 and γ2 subunits show a similar fold-increase, consistent with coregulation of the α3 and γ2 mRNAs. (D) Blocking αVβ3 integrin with LM609 or RGD prevents TGF-β1 activation and LM-α3 mRNA expression. Confluent MDCK cell monolayers were treated with either anti-αVβ3 function blocking antibody (LM609), a nonspecific antibody (IgG), RGD or RGE peptides in ExCell. After 6 h, the conditioned media were analyzed for TGF-β1 activation (gray bars), and the cells were analyzed for LM-α3 mRNA expression by qRT-PCR (black bars). n.s., not statistically significant compared with “wound”; *p < 0.05; **p < 0.01 (E) Wounded MDCK monolayers treated as described in D were analyzed by immunofluorescence for LM-332 with a polyclonal antibody (green). F-actin, red; nuclei, blue. Bar, 25 μm.

Mentions: Previous results demonstrated that TGF-β1 activation and consequent LM-332 expression occurs exclusively in subconfluent cultures of MDCK cells. Subconfluence is an in vitro phenomenon created by plating cells at low density. Epithelia in vivo are rendered “subconfluent” by wounding or injury that opens gaps in the continuous epithelium. To determine whether wounding was equivalent to subconfluence with regard to TGF-β1 activation and LM-332 expression, confluent cultures of MDCK cells were mechanically wounded by scraping multiple times with a pipette tip and analyzed. As shown in Figure 8, wounding of confluent cultures produced both an apparent increase in the total amount of TGF-β1 in the medium as well as significantly more activation (Figure 8A). Furthermore, increased LM-332 synthesis and phospho-Smad2 were evident by immunofluorescence at the wound edge (Figure 7B) and by immunoprecipitation of LM-332 after metabolic-labeling of the wounded culture (Figure 7C), suggesting that expression of LM-332 is regulated in this situation in a manner identical to that established for subconfluent cultures.


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)

Wounded confluent MDCK monolayers express LM-332. (A) Wounded epithelial monolayers activate TGF-β1. Conditioned media from a confluent MDCK cell culture (control) and a culture wounded by scraping with a pipette tip were analyzed by sandwich ELISA for total and active TGF-β1. **p < 0.01 (B) LM-332 expression is localized to the cells close to the wound edge. A confluent MDCK cell monolayer (control) and a culture injured by scraping (Wound) were analyzed after 6 h for LM-332 expression (green; left) or P-Smad2 (green/arrows; right). F-actin, red; nuclei, blue. Bar, 25 μm. (C) LM-332 synthesis is up-regulated in wounded cultures. Confluent MDCK cell monolayers (Ctrl) or wounded cultures (Wnd) were metabolically labeled with [35S]Met/Cys for 20 min 6 h after wounding, and synthesized LM-332 was analyzed by immunoprecipitation. Autoradiography revealed increased levels of α3, β3, and γ2 subunit synthesis in wounded monolayers compared with the unwounded control. The numbers between parentheses indicate fold-increase in band intensity compared with the control. Note that α3 and γ2 subunits show a similar fold-increase, consistent with coregulation of the α3 and γ2 mRNAs. (D) Blocking αVβ3 integrin with LM609 or RGD prevents TGF-β1 activation and LM-α3 mRNA expression. Confluent MDCK cell monolayers were treated with either anti-αVβ3 function blocking antibody (LM609), a nonspecific antibody (IgG), RGD or RGE peptides in ExCell. After 6 h, the conditioned media were analyzed for TGF-β1 activation (gray bars), and the cells were analyzed for LM-α3 mRNA expression by qRT-PCR (black bars). n.s., not statistically significant compared with “wound”; *p < 0.05; **p < 0.01 (E) Wounded MDCK monolayers treated as described in D were analyzed by immunofluorescence for LM-332 with a polyclonal antibody (green). F-actin, red; nuclei, blue. Bar, 25 μm.
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Figure 8: Wounded confluent MDCK monolayers express LM-332. (A) Wounded epithelial monolayers activate TGF-β1. Conditioned media from a confluent MDCK cell culture (control) and a culture wounded by scraping with a pipette tip were analyzed by sandwich ELISA for total and active TGF-β1. **p < 0.01 (B) LM-332 expression is localized to the cells close to the wound edge. A confluent MDCK cell monolayer (control) and a culture injured by scraping (Wound) were analyzed after 6 h for LM-332 expression (green; left) or P-Smad2 (green/arrows; right). F-actin, red; nuclei, blue. Bar, 25 μm. (C) LM-332 synthesis is up-regulated in wounded cultures. Confluent MDCK cell monolayers (Ctrl) or wounded cultures (Wnd) were metabolically labeled with [35S]Met/Cys for 20 min 6 h after wounding, and synthesized LM-332 was analyzed by immunoprecipitation. Autoradiography revealed increased levels of α3, β3, and γ2 subunit synthesis in wounded monolayers compared with the unwounded control. The numbers between parentheses indicate fold-increase in band intensity compared with the control. Note that α3 and γ2 subunits show a similar fold-increase, consistent with coregulation of the α3 and γ2 mRNAs. (D) Blocking αVβ3 integrin with LM609 or RGD prevents TGF-β1 activation and LM-α3 mRNA expression. Confluent MDCK cell monolayers were treated with either anti-αVβ3 function blocking antibody (LM609), a nonspecific antibody (IgG), RGD or RGE peptides in ExCell. After 6 h, the conditioned media were analyzed for TGF-β1 activation (gray bars), and the cells were analyzed for LM-α3 mRNA expression by qRT-PCR (black bars). n.s., not statistically significant compared with “wound”; *p < 0.05; **p < 0.01 (E) Wounded MDCK monolayers treated as described in D were analyzed by immunofluorescence for LM-332 with a polyclonal antibody (green). F-actin, red; nuclei, blue. Bar, 25 μm.
Mentions: Previous results demonstrated that TGF-β1 activation and consequent LM-332 expression occurs exclusively in subconfluent cultures of MDCK cells. Subconfluence is an in vitro phenomenon created by plating cells at low density. Epithelia in vivo are rendered “subconfluent” by wounding or injury that opens gaps in the continuous epithelium. To determine whether wounding was equivalent to subconfluence with regard to TGF-β1 activation and LM-332 expression, confluent cultures of MDCK cells were mechanically wounded by scraping multiple times with a pipette tip and analyzed. As shown in Figure 8, wounding of confluent cultures produced both an apparent increase in the total amount of TGF-β1 in the medium as well as significantly more activation (Figure 8A). Furthermore, increased LM-332 synthesis and phospho-Smad2 were evident by immunofluorescence at the wound edge (Figure 7B) and by immunoprecipitation of LM-332 after metabolic-labeling of the wounded culture (Figure 7C), suggesting that expression of LM-332 is regulated in this situation in a manner identical to that established for subconfluent cultures.

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