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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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MDCK cells constitutively secrete endogenous TGF-β1 but only activate it under subconfluent conditions. (A) Latent TGF-β1 is activated only in subconfluent cells. Subconfluent and confluent cultures were grown in normal growth medium (containing 5% FBS) and the conditioned media were analyzed for total and active TGF-β1 by sandwich ELISA. **p < 0.01 (B) Cells grown in ExCell also express LM-332 only when subconfluent. MDCK cells were grown in ExCell, a defined serum-free growth media, on Transwell supports for 1 d (subconfluent) or 4 d (confluent); fixed; stained; and analyzed for LM-332 expression (red) by confocal immunofluorescence microscopy; nuclei were stained with DAPI (blue). Bar, 10 μm. (C) MDCK cells constitutively secrete latent TGF-β1. Conditioned media of cells grown in ExCell and normal serum-containing growth medium (GM) were analyzed for total and active TGF-β1 using sandwich ELISA. ***p < 0.001 (D) Confluent MDCK cells secrete latent TGF-β1 apically. MDCK cells were grown in ExCell serum-free medium on Transwell supports for 4 d to achieve full apicobasal polarization. Conditioned medium from either the apical or basal compartments was analyzed for total TGF-β1 by sandwich ELISA. *p < 0.05 (E) Neutralization of endogenous active TGF-β1 reduces LM α3 subunit expression. Subconfluent MDCK cell cultures grown in ExCell in the presence of 1 μg of neutralizing antibody against active TGF-β1 (nTGF-β1) or IgG as negative control were analyzed for α3 subunit mRNA levels by qRT-PCR. *p = 0.0143.
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Figure 5: MDCK cells constitutively secrete endogenous TGF-β1 but only activate it under subconfluent conditions. (A) Latent TGF-β1 is activated only in subconfluent cells. Subconfluent and confluent cultures were grown in normal growth medium (containing 5% FBS) and the conditioned media were analyzed for total and active TGF-β1 by sandwich ELISA. **p < 0.01 (B) Cells grown in ExCell also express LM-332 only when subconfluent. MDCK cells were grown in ExCell, a defined serum-free growth media, on Transwell supports for 1 d (subconfluent) or 4 d (confluent); fixed; stained; and analyzed for LM-332 expression (red) by confocal immunofluorescence microscopy; nuclei were stained with DAPI (blue). Bar, 10 μm. (C) MDCK cells constitutively secrete latent TGF-β1. Conditioned media of cells grown in ExCell and normal serum-containing growth medium (GM) were analyzed for total and active TGF-β1 using sandwich ELISA. ***p < 0.001 (D) Confluent MDCK cells secrete latent TGF-β1 apically. MDCK cells were grown in ExCell serum-free medium on Transwell supports for 4 d to achieve full apicobasal polarization. Conditioned medium from either the apical or basal compartments was analyzed for total TGF-β1 by sandwich ELISA. *p < 0.05 (E) Neutralization of endogenous active TGF-β1 reduces LM α3 subunit expression. Subconfluent MDCK cell cultures grown in ExCell in the presence of 1 μg of neutralizing antibody against active TGF-β1 (nTGF-β1) or IgG as negative control were analyzed for α3 subunit mRNA levels by qRT-PCR. *p = 0.0143.

Mentions: To test this, serum-containing growth medium and conditioned medium from subconfluent and confluent cultures were analyzed by an ELISA capable of distinguishing active from inactive TGF-β1. As shown in Figure 5, TGF-β1 is indeed present in serum-containing MDCK cell growth medium but is almost completely inactive (Figure 5A). In contrast, conditioned medium taken from subconfluent cultures had a significant fraction of activated TGF-β1, whereas the medium from confluent cultures only contained inactive TGF-β1 (Figure 5A).


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)

MDCK cells constitutively secrete endogenous TGF-β1 but only activate it under subconfluent conditions. (A) Latent TGF-β1 is activated only in subconfluent cells. Subconfluent and confluent cultures were grown in normal growth medium (containing 5% FBS) and the conditioned media were analyzed for total and active TGF-β1 by sandwich ELISA. **p < 0.01 (B) Cells grown in ExCell also express LM-332 only when subconfluent. MDCK cells were grown in ExCell, a defined serum-free growth media, on Transwell supports for 1 d (subconfluent) or 4 d (confluent); fixed; stained; and analyzed for LM-332 expression (red) by confocal immunofluorescence microscopy; nuclei were stained with DAPI (blue). Bar, 10 μm. (C) MDCK cells constitutively secrete latent TGF-β1. Conditioned media of cells grown in ExCell and normal serum-containing growth medium (GM) were analyzed for total and active TGF-β1 using sandwich ELISA. ***p < 0.001 (D) Confluent MDCK cells secrete latent TGF-β1 apically. MDCK cells were grown in ExCell serum-free medium on Transwell supports for 4 d to achieve full apicobasal polarization. Conditioned medium from either the apical or basal compartments was analyzed for total TGF-β1 by sandwich ELISA. *p < 0.05 (E) Neutralization of endogenous active TGF-β1 reduces LM α3 subunit expression. Subconfluent MDCK cell cultures grown in ExCell in the presence of 1 μg of neutralizing antibody against active TGF-β1 (nTGF-β1) or IgG as negative control were analyzed for α3 subunit mRNA levels by qRT-PCR. *p = 0.0143.
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Figure 5: MDCK cells constitutively secrete endogenous TGF-β1 but only activate it under subconfluent conditions. (A) Latent TGF-β1 is activated only in subconfluent cells. Subconfluent and confluent cultures were grown in normal growth medium (containing 5% FBS) and the conditioned media were analyzed for total and active TGF-β1 by sandwich ELISA. **p < 0.01 (B) Cells grown in ExCell also express LM-332 only when subconfluent. MDCK cells were grown in ExCell, a defined serum-free growth media, on Transwell supports for 1 d (subconfluent) or 4 d (confluent); fixed; stained; and analyzed for LM-332 expression (red) by confocal immunofluorescence microscopy; nuclei were stained with DAPI (blue). Bar, 10 μm. (C) MDCK cells constitutively secrete latent TGF-β1. Conditioned media of cells grown in ExCell and normal serum-containing growth medium (GM) were analyzed for total and active TGF-β1 using sandwich ELISA. ***p < 0.001 (D) Confluent MDCK cells secrete latent TGF-β1 apically. MDCK cells were grown in ExCell serum-free medium on Transwell supports for 4 d to achieve full apicobasal polarization. Conditioned medium from either the apical or basal compartments was analyzed for total TGF-β1 by sandwich ELISA. *p < 0.05 (E) Neutralization of endogenous active TGF-β1 reduces LM α3 subunit expression. Subconfluent MDCK cell cultures grown in ExCell in the presence of 1 μg of neutralizing antibody against active TGF-β1 (nTGF-β1) or IgG as negative control were analyzed for α3 subunit mRNA levels by qRT-PCR. *p = 0.0143.
Mentions: To test this, serum-containing growth medium and conditioned medium from subconfluent and confluent cultures were analyzed by an ELISA capable of distinguishing active from inactive TGF-β1. As shown in Figure 5, TGF-β1 is indeed present in serum-containing MDCK cell growth medium but is almost completely inactive (Figure 5A). In contrast, conditioned medium taken from subconfluent cultures had a significant fraction of activated TGF-β1, whereas the medium from confluent cultures only contained inactive TGF-β1 (Figure 5A).

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