Figure 1.
Expression of PKD1 in normal and IPF bronchiolar epithelia.
Normal and IPF lung sections were subjected to immunohistochemical analysis by a PKD1 specific polyclonal antibody PKD1 (A-20) or a normal rabbit IgG at a dilution of 1∶500. A and A′: normal lung bronchiolar epithelium was negative for PKD1. The region indicated in panel A (magnification, ×100) is shown at higher magnification in A′ (×400). B–C′: in IPF lungs, PKD1 (red) was expressed abundantly in cilia (green arrows) and moderately in nuclei (blue arrows) of IPF BECs and in smooth muscle cells (B, black arrows). The regions indicated in panels B and C (×100) are shown at higher magnification in B′ and C′ (×400). D and D′: no specific signal was observed in IPF lung bronchiolar epithelium by a normal rabbit IgG. The region indicated in panel D (×100) is shown at higher magnification in D′ (×400).
Table 1.
Summary of PKD expression and activation in normal and IPF lung sections.
Figure 2.
Expression of PKD1 in normal and IPF alveoli.
Normal and IPF lung sections were subjected to immunohistochemical analysis by a PKD1 specific antibody PKD1 (A-20) at a dilution of 1∶500. A: normal lung AECs (black arrow) and macrophages (orange arrow) were negative for PKD1. Final magnification: ×400. B: in non-fibrotic areas of IPF lung alveoli, PKD1 (red) were expressed in the cytoplasm and nuclei of macrophages (green arrows) and AECs, including type II pneumocytes (pink arrows). Final magnification: ×400. C–D′: in the fibrotic areas of IPF lung, PKD1 (red) was expressed in flat (red arrows) and cuboidal (pink arrows) regenerative AECs lining remodeled fibrotic alveolar septa and/or fibroblast foci. Strong PKD1 immunoreactivity was also observed in macrophages (green arrows). The regions indicated in panels C and D (×100) are shown at higher magnification in C′ and D′ (×400). E and E′: PKD1 (red) were expressed in some fibroblasts or myofibroblasts (blue arrows) in fibroblasts foci of IPF lung and in regenerative AECs (pink arrows) covering the fibroblast foci as well as in macrophages (green arrow). The region indicated in panel E (×200) is shown at higher magnification in D′ (×400).
Figure 3.
Expression of PKD2 in normal and IPF lung tissues.
Normal and IPF lung sections were subjected to immunohistochemical analysis by a PKD2 specific antibody at a dilution of 1∶300. A and B: normal lung BECs and AECs (black arrow) as well as macrophages (blue arrows) were stained negative for the PKD2 antibody. Final magnification: ×400. C: in IPF bronchiolar epithelium, PKD2 (red) was expressed in the cytoplasm and nuclei of BECs (pink arrows) and in smooth muscle cells (red arrows). Final magnification: ×400. D: PKD2 (red) was expressed in macrophages (green arrows) but not neutrophils (blue arrows) in IPF lung alveoli. Final magnification: ×400. E–E3: in the fibrotic areas of IPF lung, PKD2 (red) was expressed in the flat (red arrows) and cuboidal (pink arrows) regenerative AECs lining remodeled fibrotic alveolar septa and/or fibroblast foci. It should be noted that alveolar walls grew and expanded towards the regenerative AECs overexpressing PKD2. Strong PKD2 immunoreactivity was also observed in macrophages (green arrows). The regions indicated in panel E (×100) are shown at higher magnification in E1 (×200), E2 (×400), and E3 (×400).
Figure 4.
Expression of PKD3 in normal and IPF lung tissues.
Normal and IPF lung sections were subjected to immunohistochemical analysis by a PKD3 specific antibody at a dilution of 1∶500. A and B: normal lung BECs and AECs (black arrows) were weakly stained in the nuclei by the PKD3 antibody. Final magnification: ×400. C and C′: in IPF bronchi, PKD3 (red) was expressed in the cytoplasm (pink arrows) and nuclei (green arrows) of BECs and in smooth muscle cells (C, blue arrows). The region indicated in panel C (×100) is shown at higher magnification in C′ (×400). D and D′: in the fibrotic areas of IPF lung, PKD3 (red) was expressed in the cuboidal regenerative AECs (pink arrows) lining remodeled fibrotic alveolar septa and/or fibroblast foci and in macrophages (green arrows). The region indicated in panel D (×100) is shown at higher magnification in D′ (×400).
Figure 5.
PKD family kinases are activated in bronchiolar and alveolar epithelia as well as macrophages in IPF.
Normal and IPF lung sections were subjected to immunohistochemical analysis by using a phospho-specific PKD-pSer744/748 antibody at a dilution of 1∶50. A and B: normal lung BECs, AECs, and macrophages (black arrows) were all stained negative by PKD-pSer744/748 antibody. Final magnification: ×400. C and D: positive immunoreactivities (red) were detected in the cilia (pink arrows), cytoplasm (red arrows) and nuclei (green arrows) of IPF bronchiolar (C, ×400) and honeycomb cyst (D, ×200) epithelia. Positive staining was also observed in the smooth muscle cells (C, blue arrows) surrounding small airways. E–F′: in IPF alveoli, PKD phosphorylation on Ser-744/748 (red) was readily detected in most of the hyperplastic type II pneumoctyes (E and E′, pink arrows) and the regenerative AECs (F and F′, pink arrows) lining remodeled fibrotic alveolar septa and/or fibroblast foci. It should be noted that alveolar walls grew and expanded towards the regenerative AECs with activated PKDs (F and F′). Strong positive immunoractivities (red) were also observed in alveolar macrophages (green arrows). The regions indicated in panels E and F (×100) are shown at higher magnification in E′ (×400) and F′ (×200).
Figure 6.
Agonist-induced activation of PKD in lung epithelial cells.
Primary human small airway epithelial cells (SAECs) (A) and A549 (B) cells were starved 2 h and then treated for 10 min with control vehicle, 10 ng/ml TGFβ, 5 ng/ml TNFα, 2 U/ml thrombin, 10 µM LPA, 50 ng/ml EGF, 50 ng/ml PDFDβ, 50 ng/ml FGF, 10 µM epinephrine, 25 ng/ml interlukin-6 (IL-6), 1 µM poly-L-arginine (high molecular weight: PLA-H or low molecular weight: PLA-L), 5 µg/ml CpG oligonucleotide, 10 µg/ml poly (I:C) (high molecular weight: PIC-H or low molecular weight: PIC-L), 10 µg/ml PGN, 10 µg/ml Pam3CSK4, or 1 µg/ml lipopolysaccharides (LPS). Cell lysates at equal protein amounts (45 µg) were analyzed by Western blotting with PKD-pSer744/748 or actin antibodies as indicated. Results represent western blots of three independent experiments.
Figure 7.
PKD promotes lung epithelial barrier permeability in the presence or absence of co-cultured primary lung fibroblasts or endothelial cells.
A: pooled 16HBE14o- cells stably expressing control GFP or GFP-PKD3 as described [22] were grown on Transwell inserts in DMEM medium supplemented with 7.5% FBS in the presence or absence of co-cultured primary human lung fibroblasts derived from IPF lungs (99A and 110A) and normal subjects (131N and 13N) in the bottom chamber. TEER (Ohm×cm2) was measured on day 6 using an EVOMX voltohmmeter. B: pooled 16HBE14o- cells stably expressing control GFP or GFP-PKD3 were grown on Transwell inserts in EGM-2 medium with or without co-cultured HPAECs in the bottom chamber and TEER was measured on day 5. All data are means ± S.E. (n = 3). *, p<0.05; **, p<0.01 versus control GFP control cells. Similar results were obtained in three independent experiments.