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. 2006 Jan 1;173(1):112-21.
doi: 10.1164/rccm.200507-1058OC. Epub 2005 Sep 22.

Negative regulation of myofibroblast differentiation by PTEN (Phosphatase and Tensin Homolog Deleted on chromosome 10)

Eric S White  1 , Rachelle G Atrasz, Biao Hu, Sem H Phan, Vuk Stambolic, Tak W Mak, Cory M Hogaboam, Kevin R Flaherty, Fernando J Martinez, Christopher D Kontos, Galen B Toews
Affiliations

Negative regulation of myofibroblast differentiation by PTEN (Phosphatase and Tensin Homolog Deleted on chromosome 10)

Eric S White et al. Am J Respir Crit Care Med. .

Abstract

Rationale: Myofibroblasts are primary effector cells in idiopathic pulmonary fibrosis (IPF). Defining mechanisms of myofibroblast differentiation may be critical to the development of novel therapeutic agents.

Objective: To show that myofibroblast differentiation is regulated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) activity in vivo, and to identify a potential mechanism by which this occurs.

Methods: We used tissue sections of surgical lung biopsies from patients with IPF to localize expression of PTEN and alpha-smooth muscle actin (alpha-SMA). We used cell culture of pten(-/-) and wild-type fibroblasts, as well as adenoviral strategies and pharmacologic inhibitors, to determine the mechanism by which PTEN inhibits alpha-SMA, fibroblast proliferation, and collagen production.

Results: In human lung specimens of IPF, myofibroblasts within fibroblastic foci demonstrated diminished PTEN expression. Furthermore, inhibition of PTEN in mice worsened bleomycin-induced fibrosis. In pten(-/-) fibroblasts, and in normal fibroblasts in which PTEN was inhibited, alpha-SMA, proliferation, and collagen production was upregulated. Addition of transforming growth factor-beta to wild-type cells, but not pten(-/-) cells, resulted in increased alpha-SMA expression in a time-dependent fashion. In pten(-/-) cells, reconstitution of PTEN decreased alpha-SMA expression, proliferation, and collagen production, whereas overexpression of PTEN in wild-type cells inhibited transforming growth factor-beta-induced myofibroblast differentiation. It was observed that both the protein and lipid phosphatase actions of PTEN were capable of modulating the myofibroblast phenotype.

Conclusions: The results indicate that in IPF, myofibroblasts have diminished PTEN expression. Inhibition of PTEN in vivo promotes fibrosis, and PTEN inhibits myofibroblast differentiation in vitro.

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Figures

<b>Figure 1.</b>
Figure 1.
(A) Immunohistochemical analysis of phosphatase and tensin homolog deleted on chromosome 10 (PTEN; left panel) and α–smooth muscle actin (α-SMA; right panel) in a biopsy specimen from a patient with pulmonary fibrosis. Arrows identify clusters of spindle-shaped myofibroblasts (fibroblastic foci), which are characterized by α-SMA expression and a relative decrease in PTEN expression. Arrowheads delineate cuboidal epithelial cells (which express PTEN but not α-SMA) lining distorted airspaces. Photomicrographs are representative of fibroblastic foci observed throughout the lungs of all 10 patients examined (200× original magnification). (B) Triple immunofluorescent staining of the same biopsy specimen from the patient in (A) for PTEN (fluorescein isothiocyanate, green), α-SMA (Cy3, red), and nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue). Whereas PTEN is located in epithelial and interstitial cells, it is not observed in α-SMA–expressing myofibroblasts (400× original magnification).
<b>Figure 1.</b>
Figure 1.
(A) Immunohistochemical analysis of phosphatase and tensin homolog deleted on chromosome 10 (PTEN; left panel) and α–smooth muscle actin (α-SMA; right panel) in a biopsy specimen from a patient with pulmonary fibrosis. Arrows identify clusters of spindle-shaped myofibroblasts (fibroblastic foci), which are characterized by α-SMA expression and a relative decrease in PTEN expression. Arrowheads delineate cuboidal epithelial cells (which express PTEN but not α-SMA) lining distorted airspaces. Photomicrographs are representative of fibroblastic foci observed throughout the lungs of all 10 patients examined (200× original magnification). (B) Triple immunofluorescent staining of the same biopsy specimen from the patient in (A) for PTEN (fluorescein isothiocyanate, green), α-SMA (Cy3, red), and nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue). Whereas PTEN is located in epithelial and interstitial cells, it is not observed in α-SMA–expressing myofibroblasts (400× original magnification).
<b>Figure 2.</b>
Figure 2.
Loss of pten corresponds with increased α-SMA expression in fibroblasts. (A) Western blot of whole-cell lysates derived from serum-starved wild-type murine embryonic fibroblasts (wt) or embryonic fibroblasts from pten−/− mice (pten−/−) probed for α-SMA. Blots were stripped and reprobed for β-tubulin as a loading control. The results are representative of three independent experiments using three separate cultures of cells. (B) Indirect immunofluorescent analysis of serum-starved wt fibroblasts (left panel) or pten−/− fibroblasts (right panel) labeled with anti–α-SMA antibody. Results are representative of two independent experiments. (C) The wt fibroblasts were growth-arrested and then treated with either serum-free media (SF) or 200 nM bisperoxo(pyridine-2-carboxyl)oxovanadate (bpV[pic]) for 24 h, lysed, and immunoblotted for α-SMA, phosphorylated Akt at serine-473 (S473 pAkt), and total Akt. Results are representative of three separate experiments.
<b>Figure 2.</b>
Figure 2.
Loss of pten corresponds with increased α-SMA expression in fibroblasts. (A) Western blot of whole-cell lysates derived from serum-starved wild-type murine embryonic fibroblasts (wt) or embryonic fibroblasts from pten−/− mice (pten−/−) probed for α-SMA. Blots were stripped and reprobed for β-tubulin as a loading control. The results are representative of three independent experiments using three separate cultures of cells. (B) Indirect immunofluorescent analysis of serum-starved wt fibroblasts (left panel) or pten−/− fibroblasts (right panel) labeled with anti–α-SMA antibody. Results are representative of two independent experiments. (C) The wt fibroblasts were growth-arrested and then treated with either serum-free media (SF) or 200 nM bisperoxo(pyridine-2-carboxyl)oxovanadate (bpV[pic]) for 24 h, lysed, and immunoblotted for α-SMA, phosphorylated Akt at serine-473 (S473 pAkt), and total Akt. Results are representative of three separate experiments.
<b>Figure 2.</b>
Figure 2.
Loss of pten corresponds with increased α-SMA expression in fibroblasts. (A) Western blot of whole-cell lysates derived from serum-starved wild-type murine embryonic fibroblasts (wt) or embryonic fibroblasts from pten−/− mice (pten−/−) probed for α-SMA. Blots were stripped and reprobed for β-tubulin as a loading control. The results are representative of three independent experiments using three separate cultures of cells. (B) Indirect immunofluorescent analysis of serum-starved wt fibroblasts (left panel) or pten−/− fibroblasts (right panel) labeled with anti–α-SMA antibody. Results are representative of two independent experiments. (C) The wt fibroblasts were growth-arrested and then treated with either serum-free media (SF) or 200 nM bisperoxo(pyridine-2-carboxyl)oxovanadate (bpV[pic]) for 24 h, lysed, and immunoblotted for α-SMA, phosphorylated Akt at serine-473 (S473 pAkt), and total Akt. Results are representative of three separate experiments.
<b>Figure 3.</b>
Figure 3.
Reconstitution of PTEN into pten−/− cells inhibits the myofibroblast phenotype. (A, left panel) The wt and pten−/− cells were seeded in 96-well plates and allowed to proliferate in the presence of 3H-thymidine for 24 h. 3H-thymidine incorporation was measured by scintillation counting. Right panel: After infection with adenovirus encoding full-length, active PTEN (PTEN), but not empty virus (EV), proliferation was significantly attenuated. (B, left panel) Whole-cell lysates from wt and pten−/− cells were serum-starved for 24 h before evaluation for collagen production by Sircol assay. Right panel: The pten−/− cells were untreated (U) or infected with EV or active PTEN (Ad-PTEN, PTEN) for 24 h before lysis and evaluation of collagen production by Sircol assay. (C) The pten−/− cells were untreated (U), or treated with EV or with Ad-PTEN (PTEN) for 24 h. Lysates were assessed for α-SMA expression by Western blot. Blots were stripped and probed for β-tubulin as a loading control. Subsequently, the blot was stripped and reprobed to confirm PTEN expression.
<b>Figure 3.</b>
Figure 3.
Reconstitution of PTEN into pten−/− cells inhibits the myofibroblast phenotype. (A, left panel) The wt and pten−/− cells were seeded in 96-well plates and allowed to proliferate in the presence of 3H-thymidine for 24 h. 3H-thymidine incorporation was measured by scintillation counting. Right panel: After infection with adenovirus encoding full-length, active PTEN (PTEN), but not empty virus (EV), proliferation was significantly attenuated. (B, left panel) Whole-cell lysates from wt and pten−/− cells were serum-starved for 24 h before evaluation for collagen production by Sircol assay. Right panel: The pten−/− cells were untreated (U) or infected with EV or active PTEN (Ad-PTEN, PTEN) for 24 h before lysis and evaluation of collagen production by Sircol assay. (C) The pten−/− cells were untreated (U), or treated with EV or with Ad-PTEN (PTEN) for 24 h. Lysates were assessed for α-SMA expression by Western blot. Blots were stripped and probed for β-tubulin as a loading control. Subsequently, the blot was stripped and reprobed to confirm PTEN expression.
<b>Figure 3.</b>
Figure 3.
Reconstitution of PTEN into pten−/− cells inhibits the myofibroblast phenotype. (A, left panel) The wt and pten−/− cells were seeded in 96-well plates and allowed to proliferate in the presence of 3H-thymidine for 24 h. 3H-thymidine incorporation was measured by scintillation counting. Right panel: After infection with adenovirus encoding full-length, active PTEN (PTEN), but not empty virus (EV), proliferation was significantly attenuated. (B, left panel) Whole-cell lysates from wt and pten−/− cells were serum-starved for 24 h before evaluation for collagen production by Sircol assay. Right panel: The pten−/− cells were untreated (U) or infected with EV or active PTEN (Ad-PTEN, PTEN) for 24 h before lysis and evaluation of collagen production by Sircol assay. (C) The pten−/− cells were untreated (U), or treated with EV or with Ad-PTEN (PTEN) for 24 h. Lysates were assessed for α-SMA expression by Western blot. Blots were stripped and probed for β-tubulin as a loading control. Subsequently, the blot was stripped and reprobed to confirm PTEN expression.
<b>Figure 4.</b>
Figure 4.
α-SMA expression in pten−/− cells is not dependent on autocrine TGF-β signaling or Smad activation. (A) The wt or pten−/− cells were cultured in serum-free media in the presence or absence of neutralizing antibody to TGF-β or control antibody for 24 h, and cell lysates were harvested for Western blot analysis of α-SMA. The blot was stripped and reprobed for β-tubulin as a loading control. Results are representative of two separate experiments. (B, top panel) The wt or pten−/− cells were cultured in serum-free media (SF) in the presence or absence of TGF-β (2 ng/ml) for 1 h, lysed, and assessed for phospho-Smad2 by Western blot. The membrane was stripped and reprobed with an antibody against total Smad2 to confirm equal loading. Results are representative of three separate experiments. Bottom panel: Lysates from serum-starved wt and pten−/− cells were collected and immunoblotted for total Smad7. The blot was stripped and reprobed for β-tubulin. Numbers under each lane represent densitometric ratios of Smad7 to β-tubulin.
<b>Figure 4.</b>
Figure 4.
α-SMA expression in pten−/− cells is not dependent on autocrine TGF-β signaling or Smad activation. (A) The wt or pten−/− cells were cultured in serum-free media in the presence or absence of neutralizing antibody to TGF-β or control antibody for 24 h, and cell lysates were harvested for Western blot analysis of α-SMA. The blot was stripped and reprobed for β-tubulin as a loading control. Results are representative of two separate experiments. (B, top panel) The wt or pten−/− cells were cultured in serum-free media (SF) in the presence or absence of TGF-β (2 ng/ml) for 1 h, lysed, and assessed for phospho-Smad2 by Western blot. The membrane was stripped and reprobed with an antibody against total Smad2 to confirm equal loading. Results are representative of three separate experiments. Bottom panel: Lysates from serum-starved wt and pten−/− cells were collected and immunoblotted for total Smad7. The blot was stripped and reprobed for β-tubulin. Numbers under each lane represent densitometric ratios of Smad7 to β-tubulin.
<b>Figure 4.</b>
Figure 4.
α-SMA expression in pten−/− cells is not dependent on autocrine TGF-β signaling or Smad activation. (A) The wt or pten−/− cells were cultured in serum-free media in the presence or absence of neutralizing antibody to TGF-β or control antibody for 24 h, and cell lysates were harvested for Western blot analysis of α-SMA. The blot was stripped and reprobed for β-tubulin as a loading control. Results are representative of two separate experiments. (B, top panel) The wt or pten−/− cells were cultured in serum-free media (SF) in the presence or absence of TGF-β (2 ng/ml) for 1 h, lysed, and assessed for phospho-Smad2 by Western blot. The membrane was stripped and reprobed with an antibody against total Smad2 to confirm equal loading. Results are representative of three separate experiments. Bottom panel: Lysates from serum-starved wt and pten−/− cells were collected and immunoblotted for total Smad7. The blot was stripped and reprobed for β-tubulin. Numbers under each lane represent densitometric ratios of Smad7 to β-tubulin.
<b>Figure 5.</b>
Figure 5.
Increased basal expression of α-SMA in pten−/− cells is due to increased gene expression and transcription. (A) Two separate aliquots of wt and pten−/− cells were cultured in serum-free media in the presence or absence of TGF-β (2 ng/ml) for 24 h. Whole-cell lysates were evaluated by Western blot for α-SMA expression. To verify equal protein loading, the membrane was stripped and reprobed for β-tubulin. (B) The wt and pten−/− cells were cultured in serum-free media for 24 h and RNA was isolated. α-SMA gene expression was evaluated by semiquantitative real-time PCR using glyceraldehyde phosphate dehydrogenase as an internal control. Results are pooled data from two separate experiments performed in triplicate.
<b>Figure 5.</b>
Figure 5.
Increased basal expression of α-SMA in pten−/− cells is due to increased gene expression and transcription. (A) Two separate aliquots of wt and pten−/− cells were cultured in serum-free media in the presence or absence of TGF-β (2 ng/ml) for 24 h. Whole-cell lysates were evaluated by Western blot for α-SMA expression. To verify equal protein loading, the membrane was stripped and reprobed for β-tubulin. (B) The wt and pten−/− cells were cultured in serum-free media for 24 h and RNA was isolated. α-SMA gene expression was evaluated by semiquantitative real-time PCR using glyceraldehyde phosphate dehydrogenase as an internal control. Results are pooled data from two separate experiments performed in triplicate.
<b>Figure 6.</b>
Figure 6.
Inhibition of PTEN is necessary for TGF-β–induced α-SMA expression. Wt, serum-starved fibroblasts were induced to express α-SMA with TGF-β (2 ng/ml) in the presence or absence of an adenovirus encoding full-length Ad-PTEN or empty virus alone (Ad-EV). Whole-cell lysates were assessed for α-SMA expression by Western blot. The same blot was sequentially stripped and reprobed for PTEN and β-tubulin. The results are representative of two independent experiments.
<b>Figure 7.</b>
Figure 7.
PTEN suppression of α-SMA expression involves both lipid- and protein-phosphatase activity. (A) Wt, serum-starved fibroblasts were induced to express α-SMA by treating with TGF-β (2 ng/ml) or bpV(pic) (200 nM) in the presence or absence of PP2 (10 μM) or PP3 (10 μM) for 24 h. Whole-cell lysates were prepared and evaluated by Western blot for α-SMA. The blot was stripped and reprobed for β-tubulin to confirm equal protein loading. The blot is representative of two separate experiments. (B) Wt, serum-starved fibroblasts were induced to express α-SMA by treating with TGF-β (2 ng/ml) or bpV(pic) (200 nM) in the presence or absence of the PI3K inhibitors LY294002 (50 μM) or wortmannin (50 nM) for 24 h. Whole-cell lysates were prepared and evaluated by Western blot for α-SMA. The blot was stripped and reprobed for β-tubulin to confirm equal protein loading. The blot is representative of two separate experiments.
<b>Figure 7.</b>
Figure 7.
PTEN suppression of α-SMA expression involves both lipid- and protein-phosphatase activity. (A) Wt, serum-starved fibroblasts were induced to express α-SMA by treating with TGF-β (2 ng/ml) or bpV(pic) (200 nM) in the presence or absence of PP2 (10 μM) or PP3 (10 μM) for 24 h. Whole-cell lysates were prepared and evaluated by Western blot for α-SMA. The blot was stripped and reprobed for β-tubulin to confirm equal protein loading. The blot is representative of two separate experiments. (B) Wt, serum-starved fibroblasts were induced to express α-SMA by treating with TGF-β (2 ng/ml) or bpV(pic) (200 nM) in the presence or absence of the PI3K inhibitors LY294002 (50 μM) or wortmannin (50 nM) for 24 h. Whole-cell lysates were prepared and evaluated by Western blot for α-SMA. The blot was stripped and reprobed for β-tubulin to confirm equal protein loading. The blot is representative of two separate experiments.
<b>Figure 7.</b>
Figure 7.
PTEN suppression of α-SMA expression involves both lipid- and protein-phosphatase activity. (A) Wt, serum-starved fibroblasts were induced to express α-SMA by treating with TGF-β (2 ng/ml) or bpV(pic) (200 nM) in the presence or absence of PP2 (10 μM) or PP3 (10 μM) for 24 h. Whole-cell lysates were prepared and evaluated by Western blot for α-SMA. The blot was stripped and reprobed for β-tubulin to confirm equal protein loading. The blot is representative of two separate experiments. (B) Wt, serum-starved fibroblasts were induced to express α-SMA by treating with TGF-β (2 ng/ml) or bpV(pic) (200 nM) in the presence or absence of the PI3K inhibitors LY294002 (50 μM) or wortmannin (50 nM) for 24 h. Whole-cell lysates were prepared and evaluated by Western blot for α-SMA. The blot was stripped and reprobed for β-tubulin to confirm equal protein loading. The blot is representative of two separate experiments.
<b>Figure 8.</b>
Figure 8.
Inhibition of PTEN worsens experimental fibrosis. (A) C57Bl/6 mice administered intratracheal bleomycin were treated in the presence or absence of the PTEN inhibitor bpV(pic). Animals were killed 21 d after bleomycin treatment, and lungs were assessed for total collagen. Mice treated with intratracheal saline were used as controls (n = 8 for each experimental group). The figure is representative of two separately performed experiments. (B) a and b: Lung sections from a mouse receiving intratracheal saline. c and d: Lung sections from a mouse receiving intratracheal bleomycin. e and f: Lung sections from a mouse receiving intratracheal bleomycin and intraperitoneal bpV(pic). Left panels (a, c, e): Trichrome stain. Right panels (b, d, f): Immunofluorescent stain for α-SMA (red) and nuclear DAPI stain (blue). Arrows are pointing to individual cells or clusters of myofibroblasts staining positively for α-SMA. Arrowheads identify α-SMA–expressing smooth muscle cells lining large airways. α-SMA–expressing myofibroblasts colocalize to fibrotic regions of the lung (200× original magnification).
<b>Figure 8.</b>
Figure 8.
Inhibition of PTEN worsens experimental fibrosis. (A) C57Bl/6 mice administered intratracheal bleomycin were treated in the presence or absence of the PTEN inhibitor bpV(pic). Animals were killed 21 d after bleomycin treatment, and lungs were assessed for total collagen. Mice treated with intratracheal saline were used as controls (n = 8 for each experimental group). The figure is representative of two separately performed experiments. (B) a and b: Lung sections from a mouse receiving intratracheal saline. c and d: Lung sections from a mouse receiving intratracheal bleomycin. e and f: Lung sections from a mouse receiving intratracheal bleomycin and intraperitoneal bpV(pic). Left panels (a, c, e): Trichrome stain. Right panels (b, d, f): Immunofluorescent stain for α-SMA (red) and nuclear DAPI stain (blue). Arrows are pointing to individual cells or clusters of myofibroblasts staining positively for α-SMA. Arrowheads identify α-SMA–expressing smooth muscle cells lining large airways. α-SMA–expressing myofibroblasts colocalize to fibrotic regions of the lung (200× original magnification).
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