Figure 4. Hsp90 regulates the proliferation of fibroblasts in fibrotic lesions.

(A) Primary lung fibroblasts (CD45^–Col1⁺) isolated from lung fibroblast cultures of human idiopathic pulmonary fibrosis (IPF) lungs by negative selection with anti-CD45 magnetic beads. Fibroblasts were treated with vehicle or 17-AAG (0.5 μM) for 24 hours and immunostained with antibodies against proliferating cell nuclear antigen (PCNA). Scale bars: 40 μm. (B) The number of PCNA-positive cells and total DAPI-positive cells were quantified using MetaMorph image analysis software (n = 3–4/group). Proliferation is indicated as the percentage of proliferating cells in total DAPI-positive cells. Results are cumulative of 2 independent experiments (n = 3–4/group). (C) Quantification of proliferation using the BrdU incorporation assay in lung resident fibroblasts isolated from lung cell cultures of TGF-α–transgenic (TGF-α–Tg) mice on doxycycline (Dox) for 4 weeks. Fibroblasts treated with indicated doses of 17-AAG for 48 hours from beginning of the treatment relative to vehicle is plotted. One-way ANOVA with Sidak’s multiple comparisons test was used to measure significant difference. (D) Extent of proliferation was measured using BrdU incorporation assay in lung fibroblasts isolated from lung cell cultures of TGF-α–Tg mice on Dox for 4 weeks and treated with control, Hsp90AA-specific, or Hsp90AB-specific siRNA for 72 hours. Results are cumulative of 2 independent experiments (n = 4/group). One-way ANOVA with Sidak’s multiple comparisons test was used to measure significant difference. (E) Quantification of Cdk4, Igf1, Mycn, and Sphk1 gene transcripts relative to Hprt in the lung-resident fibroblasts of TGF-α–Tg mice on Dox for 4 weeks and treated with vehicle or 17-AAG (0.2 μM) for 24 hours. Results are representative of 2 independent experiments with similar results (n = 4/group). (F) Quantification of Cdk4, Igf1, Mycn, and Sphk1 gene transcripts relative to Hprt in the lung fibroblasts of TGF-α–Tg mice on Dox for 4 weeks and treated with control, Hsp90AA-specific, or Hsp90AB-specific siRNA for 72 hours. Results are representative of 2 independent experiments (n = 3–4/group). Data shown are mean ± SEM. An unpaired 2-tailed Student’s t test was performed to measure the significance. *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.00005.