Increased expression of latent TGF-β-binding protein 4 affects the fibrotic process in scleroderma by TGF-β/SMAD signaling

Abstract

Scleroderma is a fibrosis-related disorder characterized by cutaneous and internal organ fibrosis, and excessive collagen deposition in extracellular matrix (ECM) is a major cause of fibrosis. Transforming growth factor-β (TGF-β)/SMAD signaling has a central role in the pathogenesis of fibrosis by inducing abnormal collagen accumulation in ECM, and latent TGF-β-binding protein 4 (LTBP-4) affects the secretion of latent TGF-β to ECM. A previous study indicated that bleomycin (BLM) treatment increased LTBP-4 expression in lung fibroblasts of Thy-1 knockout mice with lung fibrosis, and LTBP-4 further promoted TGF-β bioavailability as well as SMAD3 phosphorylation. However, the expression and function of LTBP-4 in human scleroderma remain unclear. We aimed to investigate the potential role of LTBP-4 in scleroderma through clinical, in vivo and in vitro studies. LTBP-4 and TGF-β expressions were significantly upregulated in systemic scleroderma (SSc) patients' plasma compared with normal controls (LTBP-4, 1,215±100.2 vs 542.8±41.7 ng/ml, P<0.0001; TGF-β, 1.5±0.2 vs 0.7±0.1 ng/ml, P=0.0031), while no significant difference was found between localized scleroderma (LSc) and normal controls. The plasma concentrations of LTBP-4 and TGF-β were even higher in SSc patients with lung fibrosis (LTBP-4, 1462± 137.3 vs 892.8±113.4 ng/ml, P=0.0037; TGF-β, 2.0±0.4 vs 0.9±0.2 ng/ml, P=0.0212) and esophagus involvement (1390±134.4 vs 940.7±127.0 ng/ml, P=0.0269; TGF-β, 1.9±0.3 vs 0.9±0.2 ng/ml, P=0.0426). The area under receiver operating characteristics (ROC) curve of LTBP-4 was 0.86. Immunohistochemistry measurement also demonstrated a higher LTBP-4 expression in sclerotic skin tissue of LSc and SSc compared with normal controls. More positive fibroblasts were also found in BLM-induced scleroderma mouse model than the saline-treated group. In in vitro studies, knockdown of LTBP-4 in SSc skin fibroblasts prominently reduced downstream COL1A1, COL1A2, and COL3A1 mRNA level by 84%, 82%, and 43%, respectively, and other fibrosis-related genes' expression were also decreased. Furthermore, extracellular TGF-β level and the SMAD2/3 phosphorylation were inhibited through LTBP-4 knockdown treatment, suggesting that the knockdown of LTBP-4 reduced the collagen expression through TGF-β/SMAD signaling pathway. Taken together, these data suggest that LTBP-4 affects fibrotic process in scleroderma, and the high expression of LTBP-4 in SSc plasma may serve as a clinical biomarker in diagnosing this disease. In addition, this study also lays the theoretical foundation for targeting LTBP-4 as treatment of scleroderma.

TGFβ-mediated expression of TGFβ-activating integrins in SSc monocytes: disturbed activation of latent TGFβ?

“…Furthermore, we observed that expression of these integrin subunits was regulated by TGFβ, as demonstrated by the use of the TGFBR1 (ALK5) kinase inhibitor SB-505124 and exogenously added hTGFβ1. Although TGFβ is widely regarded as an important driver of SSc pathophysiology [1], e.g., because it stimulates the excessive production of collagen type I by skin fibroblasts and enhances the contractile properties of these cells [10], its plasma and serum levels have been a controversial topic for many years; studies have reported increased [11][12][13], equal, and, similarly to our study, decreased levels [14,15] of TGFβ in serum and plasma of the SSc patients. Possibly, these differences can be attributed to the heterogeneity of disease course in SSc or to the heterogeneity of disease duration in our cohort.…”

“…A possible explanation for the lowered bioactivity of TGFβ we measured in the SSc serum is the increased expression of LTBP4 as was recently identified [13] or the presence of other latency conferring peptides. Alternatively, immune-related compounds present in the serum possibly modulate the abovementioned cellular context of cells in our bioassay and in this way inhibit TGFβ signaling [16].…”

DHEA-induced ovarian hyperfibrosis is mediated by TGF-β signaling pathway

“…In addition, the overexpression of TGF-β could induce activation and epithelial-mesenchymal transition (EMT) [ 19 , 20 ], which facilitate fibronectin synthesis, thus, leading to extracellular matrix (ECM) production [ 21 , 22 ]. Furthermore, TGF-β could cause the deposition of ECM and inhibit the degradation of ECM, by stimulating the secretion of growth factors that promote fibrosis, such as mesenchymal marker vimentin, and a-smooth muscle actin (a-SMA) [ 23 ].…”

MiR-3606-3p inhibits systemic sclerosis through targeting TGF-β type II receptor

“…Dermal biopsy specimens from normal controls with no history of autoimmune or other dermal disease were used as control comparators. Dermal samples were transported in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) for processing the same day as described previously [27,28]. The dermal samples were washed in 75% ethanol, phosphate buffered saline (PBS), and DMEM with 10% FBS.…”

“…A Nikon Eclipse 80i microscope (Nikon, Badhoevedorp, the Netherlands) was used to measure dermal thickness. The maximal distance between the epidermal-dermal junction and the dermal-subcutaneous fat junction in all skin sections described above was analyzed according to the method mentioned in the previous report by Jiaying Lu et al [28]. Two independent examiners performed evaluation of the sections.…”