Somatic cell plasticity and Niemann-Pick type C2 protein: fibroblast activation

Abstract

A growing body of evidence points toward activated fibroblasts, also known as myofibroblasts, as one of the leading mediators in several major human pathologies including proliferative fibrotic disorders, invasive tumor growth, rheumatoid arthritis, and atherosclerosis. Niemann-Pick Type C2 (NPC2) protein has been recently identified as a product of the second gene in NPC disease. It encodes ubiquitous, highly conserved, secretory protein with the poorly defined function. Here we show that NPC2 deficiency in human fibroblasts confers their activation. The activation phenomenon was not limited to fibroblasts as it was also observed in aortic smooth muscle cells upon silencing NPC2 gene by siRNA. More importantly, activated synovial fibroblasts isolated from patients with rheumatoid arthritis were also identified as NPC2-deficient at both the NPC2 mRNA and protein levels. The molecular mechanism responsible for activation of NPC2-null cells was shown to be a sustained phosphorylation of ERK 1/2 mitogen-activated protein kinase (MAPK), which fulfills both the sufficient and necessary fibroblast activation criteria. All of these findings highlight a novel mechanism where NPC2 by negatively regulating ERK 1/2 MAPK phosphorylation may efficiently suppress development of maladaptive tissue remodeling and inflammation.

FIGURE 1.

NPC2 deficiency confers fibroblast activation. A, expression of activated fibroblast markers as probed by real-time RT-PCR is shown. IL-6 and IL-1β secretion from non-elicited normal and NPC2-null human fibroblasts as measured by ELISA is shown. B, IL-6 and IL-1β secretion from non-elicited normal and NPC2-null human fibroblasts as measured by ELISA. Data were normalized to their respective control and are shown as the mean ± S.E. (*) depicts p ≤ 0.05 as compared with control (n = 3).

FIGURE 2.

NPC2 deficiency results in enhanced cell migration; NPC2 null human dermal fibroblasts or normal human dermal fibroblasts after NPC2 gene silencing by siRNA and aortic SMC after NPC2 gene silencing by siRNA have increased migration toward 10 ng/ml PDGF-BB. Data were normalized to their respective controls and are shown as the mean ± S.E. (*) depicts p ≤ 0.05 as compared with control (n = 3).

FIGURE 3.

Scavenger receptor expression and function in NPC2-null human fibroblasts and aortic SMC. A, scavenger receptor expression and function in NPC2-null human fibroblasts compared with normal human fibroblasts is shown. B, scavenger receptor expression in aortic SMC transfected with NPC2 siRNA is shown. C, binding of modified LDL is shown. a.u., absorbance units. D, phagocytic activity of NPC2-null fibroblasts using fluorescently labeled E. coli particles is shown. Fluorescence of non-internalized particles was quenched by toluidine blue. Data shown are the mean ± S.E. (*) depicts p ≤ 0.05 as compared with control (n = 3).

FIGURE 4.

Sustained ERK 1/2 activation in NPC2-null human fibroblasts. Total (A) and phosphorylated (activated, B) ERK1/2 were detected by Western blotting in the cytosolic (Cyt) and nuclear protein (N) fractions isolated from the wild type (WT) and NPC2-null fibroblasts. The respective proteins were labeled with the specific sets of primary/fluorescently tagged secondary antibodies and were identified on the same membrane by simultaneous dual-color scanning using the high-resolution LI-COR Odyssey near-infrared imaging system. C, a merged image of A and B demonstrates high specificity of the phospho-ERK1/2 staining. D, quantitative analysis of ERK1/2 phosphorylation in NPC2-null fibroblasts is shown. The intensities of the phosphorylated ERK1/2 bands (B) were measured using the LI-COR Odyssey Version 2.0 software and normalized to that of total ERK1/2 (A). Data are the mean ± S.E. (n = 3). The asterisk (*) depicts p ≤ 0.001 as compared with control. a.u., absorbance units.

FIGURE 5.

Constitutive RTK/GFR activation in NPC2-null fibroblasts. The RTK/GFR activation was probed by human phospho-RTK antibody array using cytosolic fractions of cell lysates isolated from NPC2-null (A) and normal (B) human skin fibroblasts. Signals from particular RTK/GFRs are shown in duplicate and numerically labeled as: 1, EGF receptor; 2, Tie-2; 3, FGF receptor 2α (FGF receptor 2 IIIc); 4, ROR2; 5, Tie-1. C, shown is a semiquantitative analysis of the duplicative signals shown in A and their respective counterparts in B. The images A and B were inverted, and intensities of particular RTK/GFRs were measured and corrected for the background using Adobe Photoshop CS4 Extended software. Data shown are the average (n = 2). a.u., absorbance units.

FIGURE 6.

Analysis of NPC2 gene expression in synovial fibroblasts isolated from patients with rheumatoid arthritis. The NPC2 mRNA transcript was measured by the real-time RT-PCR in five individual specimens designated A–E. Data normalized to the respective control and shown as the mean ± S.E. (*) depict p ≤ 0.001 as compared with control. Inset, shown is a Western blot analysis of NPC2 protein expression in activated synovial fibroblasts (specimens D and E). Control, normal skin fibroblasts. Cytoplasmic (C) and nuclear (N) protein fractions were analyzed as described under “Experimental Procedures” using the near-infrared LI-COR Odyssey imaging system and its Version 2.0 software. Molecular weight markers are shown in red on both sides of the gel. The amount of the total protein loaded per lane: Control: C, 18 ng, N, 19 ng; E: C, 23 ng, N, 27 ng; D: C, 15 ng, N, 24 ng.

FIGURE 7.

Intracellular cholesterol imaging. Cell were stained with the cholesterol probe filipin as previously described (58, 59), and images were taken on the Olympus BX41 microscope equipped with the digital camera controlled by the MagnaFire 2.1 software. A, shown are normal human skin fibroblasts. B, shown are normal human skin fibroblasts 24 h after transfection with NPC2 siRNA. C, shown are human synoviocytes derived from patients with rheumatoid arthritis. D, shown are NPC2-null human skin fibroblasts. All images were taken under the same exposure time (50 ms), and their intensities were adjusted upwards to the identical levels using Adobe Photoshop CS4 Extended Software.

FIGURE 8.

Cholesterol normalization in NPC2-null cells failed to correct their activated phenotype; real-time RT-PCR analysis of activated fibroblast markers (A) and scavenger receptors (B) in NPC2-null human skin fibroblasts. Cholesterol normalization (Normal cholesterol, ∼ 30 μg/mg of protein) was performed by incubating cells in the culture medium supplemented with the lipoprotein-deficient serum for 48 h exactly as described previously (58). Data shown are the mean ± S.E. * depicts p ≤ 0.05 as compared with control (n = 3).