Hypoxia-induced mitogenic factor modulates surfactant protein B and C expression in mouse lung

Abstract

Previous studies have demonstrated a robust pulmonary expression of hypoxia-induced mitogenic factor (HIMF) during the perinatal period, when surfactant protein (SP) synthesis begins. We hypothesized that HIMF modulates SP expression and participates in lung development and maturation. The temporal-spatial expression of HIMF, SP-B, and SP-C in developing mouse lungs was examined by immunohistochemical staining, Western blot, and RT-PCR. The expression and localization of SP-B and SP-C were investigated in mouse lungs after intratracheal instillation of HIMF in adult mice. The effects of HIMF on SP-B and SP-C transcription activity, and on mRNA degradation, were investigated in mouse lung epithelial (MLE)-12 and C10 cells using the promoter-luciferase reporter assay and actinomycin D incubation. The activation of Akt, extracellular signal-regulated kinase (ERK)1/2, and p38 mitogen-activated protein kinase was explored by Western blot. Intratracheal instillation of HIMF resulted in significant increases of SP-B and SP-C production, predominantly localized to alveolar type II cells. In MLE-12 and C10 cells, HIMF enhanced SP-B and SP-C mRNA levels in a dose-dependent manner. Meanwhile, HIMF increased transcription activity and prevented actinomycin D-facilitated SP-B and SP-C mRNA degradation in MLE-12 cells. Incubation of cells with LY294002, PD098059, or U0126 abolished HIMF-induced Akt and ERK1/2 phosphorylation and suppressed HIMF-induced SP-B and SP-C production, whereas SB203580 had no effect. These results indicate that HIMF induces SP-B and SP-C production in mouse lungs and alveolar type II-like cell lines via activations of phosphatidylinositol 3-kinase/Akt and ERK1/2 mitogen-activated protein kinase, suggesting that HIMF plays critical roles in lung development and maturation.

Figure 1.

Temporal–spatial expression of hypoxia-induced mitogenic factor (HIMF), surfactant protein (SP)-B, and SP-C in developing mouse lungs. The lungs from C57Bl/6 mice at embryonic (E) Days 15, 17, 19, postnatal (P) Days 1 and 7, and adult were harvested (n = 3 for each time point). (A) Immunohistochemical staining revealed that a robust HIMF expression was found in mouse E17 and P7 lungs, whereas the intensity of SP-B staining increased from E17, and was restricted to alveolar type II (ATII) cells and bronchiolar epithelial cells. The expression pattern of SP-C was similar to that of SP-B, detectable at E17 and restricted to the cytoplasm of epithelial cells in distal lung buds. Arrows indicate positively stained cells for SP-B, SP-C, and HIMF. Scale bars: 60 μm. Western blot with proteins from lung homogenates (B) and RT-PCR from lungs at different developmental stages (C and D) indicated that HIMF expression is closely correlated with that of SP-B and SP-C in developing lungs, especially during perinatal period. * Significant increase from mouse E15 lungs (P < 0.05). Shown is one representative experiment of three, all with similar results.

Figure 1.

Temporal–spatial expression of hypoxia-induced mitogenic factor (HIMF), surfactant protein (SP)-B, and SP-C in developing mouse lungs. The lungs from C57Bl/6 mice at embryonic (E) Days 15, 17, 19, postnatal (P) Days 1 and 7, and adult were harvested (n = 3 for each time point). (A) Immunohistochemical staining revealed that a robust HIMF expression was found in mouse E17 and P7 lungs, whereas the intensity of SP-B staining increased from E17, and was restricted to alveolar type II (ATII) cells and bronchiolar epithelial cells. The expression pattern of SP-C was similar to that of SP-B, detectable at E17 and restricted to the cytoplasm of epithelial cells in distal lung buds. Arrows indicate positively stained cells for SP-B, SP-C, and HIMF. Scale bars: 60 μm. Western blot with proteins from lung homogenates (B) and RT-PCR from lungs at different developmental stages (C and D) indicated that HIMF expression is closely correlated with that of SP-B and SP-C in developing lungs, especially during perinatal period. * Significant increase from mouse E15 lungs (P < 0.05). Shown is one representative experiment of three, all with similar results.

Figure 1.

Temporal–spatial expression of hypoxia-induced mitogenic factor (HIMF), surfactant protein (SP)-B, and SP-C in developing mouse lungs. The lungs from C57Bl/6 mice at embryonic (E) Days 15, 17, 19, postnatal (P) Days 1 and 7, and adult were harvested (n = 3 for each time point). (A) Immunohistochemical staining revealed that a robust HIMF expression was found in mouse E17 and P7 lungs, whereas the intensity of SP-B staining increased from E17, and was restricted to alveolar type II (ATII) cells and bronchiolar epithelial cells. The expression pattern of SP-C was similar to that of SP-B, detectable at E17 and restricted to the cytoplasm of epithelial cells in distal lung buds. Arrows indicate positively stained cells for SP-B, SP-C, and HIMF. Scale bars: 60 μm. Western blot with proteins from lung homogenates (B) and RT-PCR from lungs at different developmental stages (C and D) indicated that HIMF expression is closely correlated with that of SP-B and SP-C in developing lungs, especially during perinatal period. * Significant increase from mouse E15 lungs (P < 0.05). Shown is one representative experiment of three, all with similar results.

Figure 1.

Temporal–spatial expression of hypoxia-induced mitogenic factor (HIMF), surfactant protein (SP)-B, and SP-C in developing mouse lungs. The lungs from C57Bl/6 mice at embryonic (E) Days 15, 17, 19, postnatal (P) Days 1 and 7, and adult were harvested (n = 3 for each time point). (A) Immunohistochemical staining revealed that a robust HIMF expression was found in mouse E17 and P7 lungs, whereas the intensity of SP-B staining increased from E17, and was restricted to alveolar type II (ATII) cells and bronchiolar epithelial cells. The expression pattern of SP-C was similar to that of SP-B, detectable at E17 and restricted to the cytoplasm of epithelial cells in distal lung buds. Arrows indicate positively stained cells for SP-B, SP-C, and HIMF. Scale bars: 60 μm. Western blot with proteins from lung homogenates (B) and RT-PCR from lungs at different developmental stages (C and D) indicated that HIMF expression is closely correlated with that of SP-B and SP-C in developing lungs, especially during perinatal period. * Significant increase from mouse E15 lungs (P < 0.05). Shown is one representative experiment of three, all with similar results.

Figure 2.

HIMF specifically enhanced SP-B and SP-C expression in mouse lungs. Recombinant HIMF protein or BSA was intratracheally instilled into adult mouse lungs (200 ng/animal in 40 μl saline; n = 3 for each group). The vehicle controls were instilled with saline (40 μl/animal; n = 3). After 6 h, the mouse lungs were collected. (A) Results of immunohistochemical staining indicated that instillation of HIMF protein, but not BSA, resulted in a significant increase in SP-B and SP-C production, mainly located at ATII and bronchiolar epithelial cells (arrows). Scale bars: 60 μm. Western blot with proteins from lung homogenates (B) and RT-PCR from lung tissues (C) indicated that SP-B and SP-C production was specifically enhanced in HIMF- instilled, but not in BSA- or saline-instilled, mouse lungs. * Significant increase from control mouse lungs instilled with saline only (P < 0.05). Triplicate experiments were performed with essentially identical results.

Figure 2.

HIMF specifically enhanced SP-B and SP-C expression in mouse lungs. Recombinant HIMF protein or BSA was intratracheally instilled into adult mouse lungs (200 ng/animal in 40 μl saline; n = 3 for each group). The vehicle controls were instilled with saline (40 μl/animal; n = 3). After 6 h, the mouse lungs were collected. (A) Results of immunohistochemical staining indicated that instillation of HIMF protein, but not BSA, resulted in a significant increase in SP-B and SP-C production, mainly located at ATII and bronchiolar epithelial cells (arrows). Scale bars: 60 μm. Western blot with proteins from lung homogenates (B) and RT-PCR from lung tissues (C) indicated that SP-B and SP-C production was specifically enhanced in HIMF- instilled, but not in BSA- or saline-instilled, mouse lungs. * Significant increase from control mouse lungs instilled with saline only (P < 0.05). Triplicate experiments were performed with essentially identical results.

Figure 2.

HIMF specifically enhanced SP-B and SP-C expression in mouse lungs. Recombinant HIMF protein or BSA was intratracheally instilled into adult mouse lungs (200 ng/animal in 40 μl saline; n = 3 for each group). The vehicle controls were instilled with saline (40 μl/animal; n = 3). After 6 h, the mouse lungs were collected. (A) Results of immunohistochemical staining indicated that instillation of HIMF protein, but not BSA, resulted in a significant increase in SP-B and SP-C production, mainly located at ATII and bronchiolar epithelial cells (arrows). Scale bars: 60 μm. Western blot with proteins from lung homogenates (B) and RT-PCR from lung tissues (C) indicated that SP-B and SP-C production was specifically enhanced in HIMF- instilled, but not in BSA- or saline-instilled, mouse lungs. * Significant increase from control mouse lungs instilled with saline only (P < 0.05). Triplicate experiments were performed with essentially identical results.

Figure 3.

HIMF-induced SP-B and SP-C production in mouse lung epithelial cell lines MLE-12 and C10. Confluent monolayers of MLE-12 and C10 were starved with culture medium supplemented with 0.1% FBS and 2 mM L-glutamine. After 24 h, cells were treated with different concentrations of HIMF for various periods, as indicated. Semiquantitative RT-PCR was performed for HIMF mRNA expression. (A and B) Incubation of cells with 10, 20, and 40 nmol/L of HIMF protein resulted in SP-B and SP-C production in a dose-dependent manner. (C and D) Time-course study indicated that HIMF-induced SP-B and SP-C production started at 6 h, and was sustained for 24 h. * Significant increase from untreated control cells (P < 0.05). Three separated experiments were performed with essentially identical results.

Figure 3.

HIMF-induced SP-B and SP-C production in mouse lung epithelial cell lines MLE-12 and C10. Confluent monolayers of MLE-12 and C10 were starved with culture medium supplemented with 0.1% FBS and 2 mM L-glutamine. After 24 h, cells were treated with different concentrations of HIMF for various periods, as indicated. Semiquantitative RT-PCR was performed for HIMF mRNA expression. (A and B) Incubation of cells with 10, 20, and 40 nmol/L of HIMF protein resulted in SP-B and SP-C production in a dose-dependent manner. (C and D) Time-course study indicated that HIMF-induced SP-B and SP-C production started at 6 h, and was sustained for 24 h. * Significant increase from untreated control cells (P < 0.05). Three separated experiments were performed with essentially identical results.

Figure 3.

HIMF-induced SP-B and SP-C production in mouse lung epithelial cell lines MLE-12 and C10. Confluent monolayers of MLE-12 and C10 were starved with culture medium supplemented with 0.1% FBS and 2 mM L-glutamine. After 24 h, cells were treated with different concentrations of HIMF for various periods, as indicated. Semiquantitative RT-PCR was performed for HIMF mRNA expression. (A and B) Incubation of cells with 10, 20, and 40 nmol/L of HIMF protein resulted in SP-B and SP-C production in a dose-dependent manner. (C and D) Time-course study indicated that HIMF-induced SP-B and SP-C production started at 6 h, and was sustained for 24 h. * Significant increase from untreated control cells (P < 0.05). Three separated experiments were performed with essentially identical results.

Figure 3.

HIMF-induced SP-B and SP-C production in mouse lung epithelial cell lines MLE-12 and C10. Confluent monolayers of MLE-12 and C10 were starved with culture medium supplemented with 0.1% FBS and 2 mM L-glutamine. After 24 h, cells were treated with different concentrations of HIMF for various periods, as indicated. Semiquantitative RT-PCR was performed for HIMF mRNA expression. (A and B) Incubation of cells with 10, 20, and 40 nmol/L of HIMF protein resulted in SP-B and SP-C production in a dose-dependent manner. (C and D) Time-course study indicated that HIMF-induced SP-B and SP-C production started at 6 h, and was sustained for 24 h. * Significant increase from untreated control cells (P < 0.05). Three separated experiments were performed with essentially identical results.

Figure 4.

Generation of HIMF overexpressing mouse lung epithelial cells. Confluent monolayers of MLE-12 cells were transfected by HIMF cDNA or control vector with Lipofectamine 2000. Stable cell lines, MLE-HIMF and MLE-Zeo, were screened based on resistance to Zeocin (400 μg/ml). (A) Western blot from culture medium indicated that MLE-HIMF cells produce higher level of HIMF protein than their parent and transfection counterparts. (B) RT-PCR demonstrated that MLE-HIMF cells have overexpressed HIMF mRNA, and enhanced SP-B and SP-C mRNA levels compared with their parent and transfection controls. (C) Luciferase assay indicated that the promoter activity of SP-B and SP-C in MLE-HIMF cells was enhanced compared with that of their parent and transfection controls (all groups are in triplicate). * Significant increase from MLE-12 parent; ^# significant increase from transfection controls (P < 0.05). Shown is one representative experiment of three, all with similar results.

Figure 4.

Generation of HIMF overexpressing mouse lung epithelial cells. Confluent monolayers of MLE-12 cells were transfected by HIMF cDNA or control vector with Lipofectamine 2000. Stable cell lines, MLE-HIMF and MLE-Zeo, were screened based on resistance to Zeocin (400 μg/ml). (A) Western blot from culture medium indicated that MLE-HIMF cells produce higher level of HIMF protein than their parent and transfection counterparts. (B) RT-PCR demonstrated that MLE-HIMF cells have overexpressed HIMF mRNA, and enhanced SP-B and SP-C mRNA levels compared with their parent and transfection controls. (C) Luciferase assay indicated that the promoter activity of SP-B and SP-C in MLE-HIMF cells was enhanced compared with that of their parent and transfection controls (all groups are in triplicate). * Significant increase from MLE-12 parent; ^# significant increase from transfection controls (P < 0.05). Shown is one representative experiment of three, all with similar results.

Figure 4.

Generation of HIMF overexpressing mouse lung epithelial cells. Confluent monolayers of MLE-12 cells were transfected by HIMF cDNA or control vector with Lipofectamine 2000. Stable cell lines, MLE-HIMF and MLE-Zeo, were screened based on resistance to Zeocin (400 μg/ml). (A) Western blot from culture medium indicated that MLE-HIMF cells produce higher level of HIMF protein than their parent and transfection counterparts. (B) RT-PCR demonstrated that MLE-HIMF cells have overexpressed HIMF mRNA, and enhanced SP-B and SP-C mRNA levels compared with their parent and transfection controls. (C) Luciferase assay indicated that the promoter activity of SP-B and SP-C in MLE-HIMF cells was enhanced compared with that of their parent and transfection controls (all groups are in triplicate). * Significant increase from MLE-12 parent; ^# significant increase from transfection controls (P < 0.05). Shown is one representative experiment of three, all with similar results.

Figure 5.

HIMF increased both promoter activities and mRNA stability of SP-B and SP-C in mouse lung epithelial MLE-12 cells. (A) Confluent monolayers of MLE-12 were cotransfected with either pGL-SPB-Luc or pGL-SPC-Luc, and pRL-TK. After 24 h, cells were incubated with HIMF protein as indicated. Cells were then lysed, and luciferase activity was measured according to the dual-luciferase assay manual. The firefly luciferase signal was normalized to the renilla luciferase signal for each individual well. After incubation with 10–80 nmol/liter of HIMF, SP-B and SP-C promoter activity in MLE-12 cells was enhanced in a dose-dependent manner. The time-course study demonstrated that the SP-B and SP-C promoter activity induced by HIMF (20 nmol/liter) started at 6 h and persisted for 24 h. (B and C) MLE-12 cells were treated with different concentrations of HIMF, and incubated with 5 μg/ml of actinomycin D for 4 , 8, and 16 h. RT-PCR was performed, and the results indicated that HIMF prevented actinomycin D–facilitated SP-B and SP-C degradation in MLE-12 cells. * Significant increase compared with MLE-12 controls treated without HIMF (P < 0.05). All experiments were in triplicate and performed three times with similar results.

Figure 5.

HIMF increased both promoter activities and mRNA stability of SP-B and SP-C in mouse lung epithelial MLE-12 cells. (A) Confluent monolayers of MLE-12 were cotransfected with either pGL-SPB-Luc or pGL-SPC-Luc, and pRL-TK. After 24 h, cells were incubated with HIMF protein as indicated. Cells were then lysed, and luciferase activity was measured according to the dual-luciferase assay manual. The firefly luciferase signal was normalized to the renilla luciferase signal for each individual well. After incubation with 10–80 nmol/liter of HIMF, SP-B and SP-C promoter activity in MLE-12 cells was enhanced in a dose-dependent manner. The time-course study demonstrated that the SP-B and SP-C promoter activity induced by HIMF (20 nmol/liter) started at 6 h and persisted for 24 h. (B and C) MLE-12 cells were treated with different concentrations of HIMF, and incubated with 5 μg/ml of actinomycin D for 4 , 8, and 16 h. RT-PCR was performed, and the results indicated that HIMF prevented actinomycin D–facilitated SP-B and SP-C degradation in MLE-12 cells. * Significant increase compared with MLE-12 controls treated without HIMF (P < 0.05). All experiments were in triplicate and performed three times with similar results.

Figure 5.

HIMF increased both promoter activities and mRNA stability of SP-B and SP-C in mouse lung epithelial MLE-12 cells. (A) Confluent monolayers of MLE-12 were cotransfected with either pGL-SPB-Luc or pGL-SPC-Luc, and pRL-TK. After 24 h, cells were incubated with HIMF protein as indicated. Cells were then lysed, and luciferase activity was measured according to the dual-luciferase assay manual. The firefly luciferase signal was normalized to the renilla luciferase signal for each individual well. After incubation with 10–80 nmol/liter of HIMF, SP-B and SP-C promoter activity in MLE-12 cells was enhanced in a dose-dependent manner. The time-course study demonstrated that the SP-B and SP-C promoter activity induced by HIMF (20 nmol/liter) started at 6 h and persisted for 24 h. (B and C) MLE-12 cells were treated with different concentrations of HIMF, and incubated with 5 μg/ml of actinomycin D for 4 , 8, and 16 h. RT-PCR was performed, and the results indicated that HIMF prevented actinomycin D–facilitated SP-B and SP-C degradation in MLE-12 cells. * Significant increase compared with MLE-12 controls treated without HIMF (P < 0.05). All experiments were in triplicate and performed three times with similar results.

Figure 6.

Phosphatidylinositol 3-kinase (PI-3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) were involved in HIMF-induced SP-B and SP-C production. Confluent monolayers of MLE-12 and C10 were starved with medium supplemented with 0.1% FBS and 2 mM L-glutamine. After 33 h, cells were incubated in serum-free medium for 3–4 h at 37°C. Cells were then pretreated with different signal transduction inhibitors for 1 h, and then stimulated with HIMF protein for various periods, as indicated. (A) Western blot with proteins from cell lysate indicated that HIMF strongly activated Akt phosphorylation at Ser473 and Thr308, and phosphorylation of ERK1/2 and p38 MAPK. (B and C) The PI-3K inhibitor LY294002 (10 μmol/liter) inhibited HIMF-activated Akt phosphorylation. Moreover, incubation of cells with SB203580, inhibitor against p38 (5 μmol/liter), and either PD098059 (5 μmol/liter) or U0126 (5 μmol/liter), inhibitors of ERK1/2 MAPK pathways, also blocked HIMF-induced phosphorylation of p38 and ERK1/2. (D) Luciferase assay revealed that HIMF-induced SP-B and SP-C expression was not abolished by p38 inhibitor SB203580. However, incubation of MLE-12 cells with the PI-3K inhibitor, LY294002, prevented HIMF-induced upregulation of both SP-B and SP-C. Meanwhile, the inhibitors of ERK1/2 MAPK, PD098059 and U0126, also blocked HIMF-induced SP-B and SP-C production, respectively, which was further confirmed by RT-PCR (E). * Significant increase compared with MLE-12 controls treated without HIMF (P < 0.05). Three experiments with triplicate for each treatment were performed, and all with similar results.

Figure 6.

Phosphatidylinositol 3-kinase (PI-3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) were involved in HIMF-induced SP-B and SP-C production. Confluent monolayers of MLE-12 and C10 were starved with medium supplemented with 0.1% FBS and 2 mM L-glutamine. After 33 h, cells were incubated in serum-free medium for 3–4 h at 37°C. Cells were then pretreated with different signal transduction inhibitors for 1 h, and then stimulated with HIMF protein for various periods, as indicated. (A) Western blot with proteins from cell lysate indicated that HIMF strongly activated Akt phosphorylation at Ser473 and Thr308, and phosphorylation of ERK1/2 and p38 MAPK. (B and C) The PI-3K inhibitor LY294002 (10 μmol/liter) inhibited HIMF-activated Akt phosphorylation. Moreover, incubation of cells with SB203580, inhibitor against p38 (5 μmol/liter), and either PD098059 (5 μmol/liter) or U0126 (5 μmol/liter), inhibitors of ERK1/2 MAPK pathways, also blocked HIMF-induced phosphorylation of p38 and ERK1/2. (D) Luciferase assay revealed that HIMF-induced SP-B and SP-C expression was not abolished by p38 inhibitor SB203580. However, incubation of MLE-12 cells with the PI-3K inhibitor, LY294002, prevented HIMF-induced upregulation of both SP-B and SP-C. Meanwhile, the inhibitors of ERK1/2 MAPK, PD098059 and U0126, also blocked HIMF-induced SP-B and SP-C production, respectively, which was further confirmed by RT-PCR (E). * Significant increase compared with MLE-12 controls treated without HIMF (P < 0.05). Three experiments with triplicate for each treatment were performed, and all with similar results.

Figure 6.

Phosphatidylinositol 3-kinase (PI-3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) were involved in HIMF-induced SP-B and SP-C production. Confluent monolayers of MLE-12 and C10 were starved with medium supplemented with 0.1% FBS and 2 mM L-glutamine. After 33 h, cells were incubated in serum-free medium for 3–4 h at 37°C. Cells were then pretreated with different signal transduction inhibitors for 1 h, and then stimulated with HIMF protein for various periods, as indicated. (A) Western blot with proteins from cell lysate indicated that HIMF strongly activated Akt phosphorylation at Ser473 and Thr308, and phosphorylation of ERK1/2 and p38 MAPK. (B and C) The PI-3K inhibitor LY294002 (10 μmol/liter) inhibited HIMF-activated Akt phosphorylation. Moreover, incubation of cells with SB203580, inhibitor against p38 (5 μmol/liter), and either PD098059 (5 μmol/liter) or U0126 (5 μmol/liter), inhibitors of ERK1/2 MAPK pathways, also blocked HIMF-induced phosphorylation of p38 and ERK1/2. (D) Luciferase assay revealed that HIMF-induced SP-B and SP-C expression was not abolished by p38 inhibitor SB203580. However, incubation of MLE-12 cells with the PI-3K inhibitor, LY294002, prevented HIMF-induced upregulation of both SP-B and SP-C. Meanwhile, the inhibitors of ERK1/2 MAPK, PD098059 and U0126, also blocked HIMF-induced SP-B and SP-C production, respectively, which was further confirmed by RT-PCR (E). * Significant increase compared with MLE-12 controls treated without HIMF (P < 0.05). Three experiments with triplicate for each treatment were performed, and all with similar results.

Figure 6.

Phosphatidylinositol 3-kinase (PI-3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) were involved in HIMF-induced SP-B and SP-C production. Confluent monolayers of MLE-12 and C10 were starved with medium supplemented with 0.1% FBS and 2 mM L-glutamine. After 33 h, cells were incubated in serum-free medium for 3–4 h at 37°C. Cells were then pretreated with different signal transduction inhibitors for 1 h, and then stimulated with HIMF protein for various periods, as indicated. (A) Western blot with proteins from cell lysate indicated that HIMF strongly activated Akt phosphorylation at Ser473 and Thr308, and phosphorylation of ERK1/2 and p38 MAPK. (B and C) The PI-3K inhibitor LY294002 (10 μmol/liter) inhibited HIMF-activated Akt phosphorylation. Moreover, incubation of cells with SB203580, inhibitor against p38 (5 μmol/liter), and either PD098059 (5 μmol/liter) or U0126 (5 μmol/liter), inhibitors of ERK1/2 MAPK pathways, also blocked HIMF-induced phosphorylation of p38 and ERK1/2. (D) Luciferase assay revealed that HIMF-induced SP-B and SP-C expression was not abolished by p38 inhibitor SB203580. However, incubation of MLE-12 cells with the PI-3K inhibitor, LY294002, prevented HIMF-induced upregulation of both SP-B and SP-C. Meanwhile, the inhibitors of ERK1/2 MAPK, PD098059 and U0126, also blocked HIMF-induced SP-B and SP-C production, respectively, which was further confirmed by RT-PCR (E). * Significant increase compared with MLE-12 controls treated without HIMF (P < 0.05). Three experiments with triplicate for each treatment were performed, and all with similar results.

Figure 6.

Phosphatidylinositol 3-kinase (PI-3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) were involved in HIMF-induced SP-B and SP-C production. Confluent monolayers of MLE-12 and C10 were starved with medium supplemented with 0.1% FBS and 2 mM L-glutamine. After 33 h, cells were incubated in serum-free medium for 3–4 h at 37°C. Cells were then pretreated with different signal transduction inhibitors for 1 h, and then stimulated with HIMF protein for various periods, as indicated. (A) Western blot with proteins from cell lysate indicated that HIMF strongly activated Akt phosphorylation at Ser473 and Thr308, and phosphorylation of ERK1/2 and p38 MAPK. (B and C) The PI-3K inhibitor LY294002 (10 μmol/liter) inhibited HIMF-activated Akt phosphorylation. Moreover, incubation of cells with SB203580, inhibitor against p38 (5 μmol/liter), and either PD098059 (5 μmol/liter) or U0126 (5 μmol/liter), inhibitors of ERK1/2 MAPK pathways, also blocked HIMF-induced phosphorylation of p38 and ERK1/2. (D) Luciferase assay revealed that HIMF-induced SP-B and SP-C expression was not abolished by p38 inhibitor SB203580. However, incubation of MLE-12 cells with the PI-3K inhibitor, LY294002, prevented HIMF-induced upregulation of both SP-B and SP-C. Meanwhile, the inhibitors of ERK1/2 MAPK, PD098059 and U0126, also blocked HIMF-induced SP-B and SP-C production, respectively, which was further confirmed by RT-PCR (E). * Significant increase compared with MLE-12 controls treated without HIMF (P < 0.05). Three experiments with triplicate for each treatment were performed, and all with similar results.