The role of glycoprotein 130 family of cytokines in fetal rat lung development

Abstract

The glycoprotein 130 (gp130) dependent family of cytokines comprises interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), cardiotrophin-like cytokine (CLC), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1) and oncostatin M (OSM). These cytokines share the membrane gp130 as a common signal transducer. Recently, it was demonstrated that IL-6 promotes, whereas LIF inhibits fetal lung branching. Thus, in this study, the effects on fetal lung morphogenesis of the other classical members of the gp130-type cytokines (IL-11, CLC, CNTF, CT-1 and OSM) were investigated. We also provide the first description of these cytokines and their common gp130 receptor protein expression patterns during rat lung development. Fetal rat lung explants were cultured in vitro with increasing concentrations of IL-11, CLC, CNTF, CT-1 and OSM. Treated lung explants were morphometrically analyzed and assessed for MAPK, PI3K/AKT and STAT3 signaling modifications. IL-11, which similarly to IL-6 acts through a gp130 homodimer receptor, significantly stimulated lung growth via p38 phosphorylation. On the other hand, CLC, CNTF, CT-1 and OSM, whose receptors are gp130 heterodimers, inhibited lung growth acting in different signal-transducing pathways. Thus, the present study demonstrated that although cytokines of the gp130 family share a common signal transducer, there are specific biological activities for each cytokine on lung development. Indeed, cytokine signaling through gp130 homodimers stimulate, whereas cytokine signaling through gp130 heterodimers inhibit lung branching.

Figure 1. IL-11 expression pattern during fetal lung development.

IL-11 is expressed throughout all stages of lung development studied, from early 13.5 dpc until late 21.5 dpc and also in the adult. Representative immunohistochemistry staining of (A–E) developing lung and (F) adult lung. Original magnification ×100.

Figure 2. CLC expression pattern during fetal lung development.

CLC is expressed throughout all stages of lung development studied, from early 13.5 dpc until late 21.5 dpc and also in the adult. Representative immunohistochemistry staining of (A–E) developing lung and (F) adult lung. Original magnification ×100.

Figure 3. CNTF expression pattern during fetal lung development.

CNTF is expressed throughout all stages of lung development studied, from early 13.5 dpc until late 21.5 dpc and also in the adult. Representative immunohistochemistry staining of (A–E) developing lung and (F) adult lung. Original magnification ×100.

Figure 4. CT-1 expression pattern during fetal lung development.

CT-1 is expressed throughout all stages of lung development studied, from early 13.5 dpc until late 21.5 dpc and also in the adult. Representative immunohistochemistry staining of (A–E) developing lung and (F) adult lung. Original magnification ×100.

Figure 5. OSM expression pattern during fetal lung development.

OSM is expressed throughout all stages of lung development studied, from early 13.5 dpc until late 21.5 dpc and also in the adult. Representative immunohistochemistry staining of (A–E) developing lung and (F) adult lung. Original magnification ×100.

Figure 6. Gp130 receptor expression pattern during fetal lung development.

Gp130 receptor is expressed throughout all stages of lung development studied, from early 13.5 dpc until late 21.5 dpc and also in the adult. Representative immunohistochemistry staining of (A–E) developing lung and (F) adult lung. Original magnification ×100.

Figure 7. IL-11 supplementation studies in a fetal lung explant culture system.

(A) Representative examples of fetal lung explants treated daily with increasing concentrations of recombinant IL-11, after 4 days in culture. Original magnification: ×25. (B) Number of peripheral airway buds ; (C) Epithelial perimeter; (D) Area; (E) External perimeter of lung explants treated with IL-11. Results are expressed as ratio of day 4 (D₄) and day 0 (D₀) of culture (D₄/D₀ ratio), and also as absolute number in D₀ (black bar) and D₄ (white bar). p<0.05: ^* vs. IL-11 at 0 pg/mL (control), ^§ vs. IL-11 at 0.1 pg/mL, ^¥ vs. IL-11 at 1 pg/mL.

Figure 8. CLC supplementation studies in a fetal lung explant culture system.

(A) Representative examples of fetal lung explants treated daily with increasing concentrations of recombinant CLC, after 4 days in culture. Original magnification: ×25. (B) Number of peripheral airway buds; (C) Epithelial perimeter; (D) Area; (E) External perimeter of lung explants treated with CLC. Results are expressed as ratio of day 4 (D₄) and day 0 (D₀) of culture (D₄/D₀ ratio), and also as absolute number in D₀ (black bar) and D₄ (white bar). p<0.05: ^* vs. CLC at 0 nM (control plus 4 mM HCl), ^§ vs. CLC at 0.003 nM, ^¥ vs. CLC at 0.03 nM, † vs. CLC at 0.3 nM, ‡ vs. CLC at 3 nM.

Figure 9. CNTF supplementation studies in a fetal lung explant culture system.

(A) Representative examples of fetal lung explants treated daily with increasing concentrations of recombinant CNTF, after 4 days in culture. Original magnification: ×25. (B) Number of peripheral airway buds; (C) Epithelial perimeter; (D) Area; (E) External perimeter of lung explants treated with CNTF. Results are expressed as ratio of day 4 (D₄) and day 0 (D₀) of culture (D₄/D₀ ratio), and also as absolute number in D₀ (black bar) and D₄ (white bar). p<0.05: ^* vs. CNTF at 0 nM (control), ^§ vs. CNTF at 0.1 ng/mL, ^¥ vs. CNTF at 1 ng/mL, † vs. CNTF at 10 ng/mL.

Figure 10. CT-1 supplementation studies in a fetal lung explant culture system.

(A) Representative examples of fetal lung explants treated daily with increasing concentrations of recombinant CT-1, after 4 days in culture. Original magnification: ×25. (B) Number of peripheral airway buds; (C) Epithelial perimeter; (D) Area; (E) External perimeter of lung explants treated with CT-1. Results are expressed as ratio of day 4 (D₄) and day 0 (D₀) of culture (D₄/D₀ ratio), and also as absolute number in D₀ (black bar) and D₄ (white bar). p<0.05: ^* vs. CT-1 at 0 ng/mL (control plus 4 mM HCl), ^§ vs. CT-1 at 0.1 ng/mL, ^¥ vs. CT-1 at 1 ng/mL, † vs. CT-1 at 10 ng/mL, ‡ vs. CT-1 at 100 ng/mL.

Figure 11. OSM supplementation studies in a fetal lung explant culture system.

(A) Representative examples of fetal lung explants treated daily with increasing concentrations of recombinant OSM, after 4 days in culture. Original magnification: ×25. (B) Number of peripheral airway buds; (C) Epithelial perimeter; (D) Area; (E) External perimeter of lung explants treated with OSM. Results are expressed as ratio of day 4 (D₄) and day 0 (D₀) of culture (D₄/D₀ ratio), and also as absolute number in D₀ (black bar) and D₄ (white bar). p<0.05: ^* vs. OSM at 0 ng/mL (control), † vs. OSM at 0.1 ng/mL, ^§ vs. OSM at 1 ng/mL, ^¥ vs. OSM at 10 ng/mL.

Figure 12. Analysis of intracellular signaling pathways that IL-11, CLC, CNTF, CT-1 and OSM supplementation mediates on lung growth.

(A) Western blot analysis of p38, p44/42, JNK1/2, AKT, STAT3 and SOCS3, and also diphosphorylated forms of p38 (dp-p38), p44/42 (dp-p44/42), SAPK/JNK (dp-JNK1/2), AKT (dp-AKT) and STAT3 (dp-STAT3) in control (1), control plus 4 mM HCl for CLC and CT-1 lung explants (2) and treated with IL-11 at 0.1 pg/mL (3), CLC at 30 nM (4), CNTF at 1000 ng/mL (5), CT-1 at 200 ng/mL (6), and OSM at 100 ng/mL (7). Control loading was performed using β-tubulin (55 kDa). p38 corresponds to 38 kDa. p44/42 corresponds to 44 and 42 kDa, respectively. JNK1 and 2 corresponds to 46 and 54 kDa, respectively. AKT corresponds to 60 kDa. STAT3 corresponds to two bands, 79 and 86 kDa. SOCS3 corresponds to 30 kDa. Semi-quantitative analysis for (B) dp-p38, (C) dp-p44/42, (D) dp-JNK1/2, (E) dp-AKT, (F) dp-STAT3, and (G) SOCS3. Results are presented as arbitrary units normalized for β-tubulin and the respective control. p<0.05: ^* vs. control.

Figure 13. Effects of IL-11, CLC, CNTF, CT-1 and OSM supplementation on proliferation and apoptosis.

(A) Western blot analysis of PCNA and cleaved PARP in control (1), control plus 4 mM HCl for CLC and CT-1 lung explants (2) and treated with IL-11 at 0.1 pg/mL (3), CLC at 30 nM (4), CNTF at 1000 ng/mL (5), CT-1 at 200 ng/mL (6), and OSM at 100 ng/mL (7). Control loading was performed using β-tubulin (55 kDa). PCNA corresponds to 36 kDa. Cleaved PARP corresponds to 89 kDa. Semi-quantitative analysis for (B) PCNA, p = 0.003, ^* vs. control and (C) cleaved PARP, p<0.05: ^* vs. control. Results are presented as arbitrary units normalized for β-tubulin and the respective control.

Figure 14. Overview of the role of gp130 family of cytokines in fetal lung development, cytokines signaling through gp130 homodimers (IL-6 and IL-11) stimulate fetal lung growth, whereas cytokines acting through a gp130 heterodimer receptor (LIF, CT-1, CNTF, CLC and OSM) inhibit lung growth.