Insulin-like growth factor-I stimulates differentiation of ATII cells to ATI-like cells through activation of Wnt5a - PubMed (original) (raw)

Insulin-like growth factor-I stimulates differentiation of ATII cells to ATI-like cells through activation of Wnt5a

Manik C Ghosh et al. Am J Physiol Lung Cell Mol Physiol. 2013.

Abstract

Alveolar type II (ATII) epithelial cells play a crucial role in the repair and remodeling of the lung following injury. ATII cells have the capability to proliferate and differentiate into alveolar type I (ATI) cells in vivo and into an ATI-like phenotype in vitro. While previous reports indicate that the differentiation of ATII cells into ATI cells is a complex biological process, the underlying mechanism responsible for differentiation is not fully understood. To investigate factors involved in this differentiation in culture, we used a PCR array and identified several genes that were either up- or downregulated in ATI-like cells (day 6 in culture) compared with day 2 ATII cells. Insulin-like growth factor-I (IGF-I) mRNA was increased nearly eightfold. We found that IGF-I was increased in the culture media of ATI-like cells and demonstrated a significant role in the differentiation process. Treatment of ATII cells with recombinant IGF-I accelerated the differentiation process, and this effect was abrogated by the IGF-I receptor blocker PQ401. We found that Wnt5a, a member of the Wnt-Frizzled pathway, was activated during IGF-I-mediated differentiation. Both protein kinase C and β-catenin were transiently activated during transdifferentiation. Knocking down Wnt5a using small-interfering RNA abrogated the differentiation process as indicated by changes in the expression of an ATII cell marker (prosurfactant protein-C). Treatment of wounded cells with either IGF-I or Wnt5a stimulated wound closure. These results suggest that IGF-I promotes differentiation of ATII to ATI cells through the activation of a noncanonical Wnt pathway.

Keywords: alveolar epithelial cell; alveolar type I; alveolar type II; prosurfactant protein-C.

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Figures

Fig. 1.

Fig. 1.

Alveolar type II (ATII) cells differentiate to alveolar type I (ATI)-like cells in vitro. ATII cells were isolated from rats and cultured for 2 or 6 days. Surface markers of ATII (rTII70) and ATI (rTI40) were immunoblotted from the lysate (A and B, respectively). β-Actin was used as a loading control. Representative immunoblots and densitometry from three different isolations are shown. *Value was significant at P < 0.05 level compared with day 2 (n = 3).

Fig. 2.

Fig. 2.

Insulin-like growth factor-I (IGF-I) was upregulated during differentiation of ATII to ATI-like cells. A: mRNA was collected from ATII (day 2) and ATI-like (day 6) cells and converted to cDNA using an RT-PCR kit. PCR Array was run according to the procedure described in materials and methods. Values indicate the fold change relative to day 2 ATII cells (n = 3). B: IGF-I was secreted from wounded and unwounded ATII and ATI-like cells. Multiple scratch wounds were used to increase the percentage of cells near a wound edge. IGF-I was determined by ELISA. *Significant difference from unwounded cells; #significant difference from day 2 ATII cells (P < 0.05, n = 3–9). C: phosphorylation of insulin receptor substrate-1 was examined using the Luminex system. ATII cells (day 2) were treated with IGF-I (50 ng/ml) for the indicated times, and lysate was collected. *Significant difference from untreated cells (P < 0.05; n = 6).

Fig. 3.

Fig. 3.

Activation of Wnt3a and Wnt5a during transdifferentiation. Representative immunoblots of Wnt3a, Wnt5a, and prosurfactant protein-C (pro-SPC) from lysate from ATII cells cultured from 0 to 6 days are shown (A, B, and C, respectively). β-Actin was immunoblotted as a loading control. D: densitometry for Wnt3a, Wnt5a, and Pro-SPC (n = 4). *Significant difference from day 0 (P < 0.05). E and F: representative immunoblots and densitometry of Wnt5a (n = 5) and pro-SPC (n = 3) in A549 cells treated with IGF-I for 72 h. *Significant difference from untreated control (P < 0.05).

Fig. 4.

Fig. 4.

Activation of protein kinase C (PKC) and β-catenin during transdifferentiation. Representative immunoblots of pan-PKC and β-catenin from lysate from ATII cells cultured from 0 to 6 days are shown (A and B). β-Actin was immunoblotted as a loading control. Bottom: densitometry for pan-PKC (n = 4) and β-catenin (n = 3). *Significant difference from day 0 (P < 0.05).

Fig. 5.

Fig. 5.

Wnt5a activation was stimulated by IGF-I. ATII cells (day 2) were treated with IGF-I (50 ng/ml), PQ401 (500 ng/ml), or the combination for 24 h, and lysates were collected. Representative immunoblots of Wnt5a and Wnt3a are shown (A and B) along with densitometry for Wnt5a (n = 4) and Wnt3a (n = 4). *Significant difference from untreated controls (P < 0.05). NS, values were not significant compared with control.

Fig. 6.

Fig. 6.

Knockdown of Wnt5a by small-interfering RNA (siRNA) increased the expression of pro-SPC. Wnt5a was knocked down by siRNA in ATII cells according to the procedure described in materials and methods. A: immunoblot of Wnt5a from lysates from 4 different isolations with corresponding densitometry. β-Actin was used as a loading control. *Significant difference from cells transfected with control siRNA (P < 0.05). B: expression of pro-SPC was examined by immunofluorescence in control and Wnt5a siRNA-transfected cells. Mean fluorescence intensity was measured in 5 different fields from 5 different experiments. *Significant difference from cells transfected with control siRNA (P < 0.05; n = 5); NS, values were not significantly different from control siRNA; #significant difference from cells transfected with Wnt5a siRNA.

Fig. 7.

Fig. 7.

Wounding stimulated Wnt5a expression, and IGF-I and Wnt5a augmented wound healing in MLE-12 cells. A: MLE-12 cells were either unwounded (control) or wounded with multiple scratches, and Wnt5a expression was determined from cell lysates after 36 h. A representative immunoblot is shown, and densitometry is summarized. *Significant difference from unwounded control (P < 0.05, n = 3). B: MLE-12 cells were wounded and then treated with either murine IGF-I (50 ng/ml), murine recombinant Wnt5a (100 ng/ml), or the combination of the two. IGF-I and Wnt5a were added at time 0 and at 24 h, and wound width measurements were made at the initial time and after 36 h. *Significant difference compared with control (P < 0.05; n = 6).

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