Figure 5.

IGFBP‐4 downregulates CXCR4 and CXCR4 levels are elevated in SSc lung fibroblasts. (A) IGFBP‐4 inhibits baseline and TGF‐ß–induced CXCR4 mRNA expression. Primary human lung fibroblasts were treated with 2 µg/mL rhIGFBP‐4 or vehicle control with or without 10 ng/mL TGF‐ß1 for 24 hours. Total RNA was extracted and used for the measurement of CXCR4 mRNA levels by real‐time PCR. Data shown is from three independent experiments using fibroblasts from three different donors. (B) IGFBP‐4 reduces protein levels of CXCR4. Primary human lung fibroblasts were treated with 2 µg/mL rhIGFBP‐4 with or without 10 ng/mL TGF‐ß1 as described in (A). Cellular membrane fractions were analyzed by western blot. Experiments were repeated using lung fibroblasts from three different donors with similar results. (C) Silencing IGFBP‐4 increases CXCR4 expression. Primary human lung fibroblasts were transfected with siRNA targeting IGFBP‐4 or control scrambled siRNA for 24 hours. CXCR4 mRNA levels were measured by real time PCR. The graph summarizes results from three experiments using fibroblasts from three different donors. (D) CXCR4 mRNA levels are higher in SSc lung fibroblasts compared to normal control lung fibroblasts. CXCR4 mRNA levels were measured in fibroblasts from lung tissues of nine SSc patients and nine controls using real time PCR. *P < 0.05, **P < 0.01, ***P < 0.0001