The Effect Of ITGB4BP On The Biological Function Of Scar-Derived Fibroblasts And The Underlying Mechanisms

Hypertrophic scar, a unique feature in human, is a pathologic wound healing response after thermal injuries or traumas. Clinically, it present as erythematous, firm, elevated plaques that remain confined to the area damaged by the initial injury. Although it does not pose a health risk, some scars may be associated with pruritus, pain, disfigurement, disfunction, and psychological distress. Despite advances in burn care and rehabilitaition, as well as progress in the management during these decades, pathologic scar remains to be hard cured following surgical methods and drugs of treatment. On this background we are looking forward to finding the multitude of possible traumatic mechanisms and the underlying molecular signal ways in the formation of hypertrophic scar.The formation of hypertrophic scar represents a dysregulated response to cutaneous wound healing and is characterized by inflammation, excessive proliferation of fibroblasts, and abnormal deposition of extracellular matrix （ECM） proteins. Myofibroblasts, a type of cell differentiated from quiescent fibroblasts in a process that requies transforming growth factor-β1 （TGF-β1） and mechanical stress, have been demonstrated by many studies to play an essential role in induction and maintenance of the hypertrophic scar. While myofibroblast differentiation during normal acute wound healing is temporally limited, while, in hypertrophic scar these myofibroblasts persist in high numbers for long periods following injury. The activation of fibroblasts and myofibroblasts promote the synthesis ofα-smooth muscle actin （α-SMA）, TGF-β1, and the excessive ECM （mainly collagen I）, with enhanced the contractile ability.We previously identified p311 gene highly expressed in hypertrophic scar by gene chip. To explore the mechanism of P311 in hypertrophic scar, we found a potential interaction protein of P311 by both yeast two-hybrid system and Fluorescence Resonance Energy Transfer （FRET） screening, named integrinβ4 binding protein （ITGB4BP）. ITGB4BP (also named as eIF6 and p27^BBP) binds to the fibronectin type III domains of ITGB4 and may help link ITGB4 to the intermediate filament cytoskeleton. This encoded protein, which is insoluble and found both in the nucleus and in the cytoplasm, behaves as a ribosome-anti-association factor in nucleolus which prevents the interaction of 40S ribosomal subunits with 60S subunits through its binding to 60S ribosomes and is involved in miRNA-mediatied gene silencing in cytoplasm. Moreover, ITGB4BP does regulate Wnt signaling throughβ-catenin which might affect the cystoskeleton and adherence of cells. However, the potential roles of ITGB4BP in fibroblast biological function and in wound healing have yet to be clarified. Therefore, current study will focus on:1. Detection of ITGB4BP expression and distribution in different tissues.2. Determination of the effect of ITGB4BP on hypertrophic scar-derived fibroblast function in vitro.3. Observation of the role of ITGB4BP in the fibrosis of cutaneous incisional wound in vivo.Results:1. ITGB4BP was downregulated in hypertrophic scar-derived fibroblast The primary fibroblasts from hypertrophic scar and normal skin from the same patient were cultured to examine the expression of ITGB4BP in the fibroblasts. By means of Real-Time PCR and Western Blotting, the expression of ITGB4BP was downregulated in hypertrophic scar-derived fibroblasts both at mRNA and protein levels, as compared with that in normal skin derived-fibroblasts.Meanwhile we observed the expression of ITGB4BP in the adult normal skin, and the skin from hypertrophic scar. The immunohistochemical staining showed that ITGB4BP was positive in cytoplasm of fibroblasts in mature skin tissue, and negative in the hypertrophic scar dermal tissue, which indicate the important role of ITGB4BP in control of hypertrophic scar formation.2. ITGB4BP inhibited the myofibroblast functions The effects of transduction of ITGB4BP and its empty control adenovirus into the hypertrophic scar-derived fibroblasts were examined by means of Real-Time PCR, Western Blotting, and FPCL（Fibroblasts Populated Collagen Lattice） model. We firstly observed that the fibroblasts constitutively expressα-SMA, TGF-β1, and Collagen I. Meanwhile the upregulation of ITGB4BP did decrease the expression ofα-SMA, TGF-β1, and Collagen I.As the ability to induce collagen contraction is a characteristic feature of myofibroblasts, we detected the contractile ability of fibroblast in the FPCL model following the upregulation of ITGB4BP. The transduction of ITGB4BP significantly suppressed the ability to contract the gels.In addition, we blocked the TGF-β1 signal pathway by adding the antibody of TGF-β1 into the culture medium. The contractile ability of fibroblasts was still inhibited by ITGB4BP in FPCL model which indicated that ITGB4BP suppressed the contraction in the manner of TGF-β1 independent pathway, through the direct interaction betweenα3-Catenin and ITGB4BP, as demonstrated by means of immunoprecipetation and mass spectrometry.3. ITGB4BP suppressed the fibrosis in the animal models We further observed the effect of overexpression of ITGB4BP on wound fibrosis in a full thickness incisional wound healing model and in the liver fibrosis model in rats. The results displayed that ITGB4BP could decrease the area of scar in the incisional wound after 7 days injury, and suppress to some extends the development of liver fibrosis in rats.In summary, we for the first time demonstrated that ITGB4BP was involved in the fibroblast/myofibroblast biological function, myofibroblast de-differentiation and tissue fibrosis in either TGF-β1 dependent or TGF-β1 independent pathway （i.e.,β-Catenin signal pathway）. Our data strongly suggested that ITGB4BP might be a new potential target to control the fibrosis and hypertrophic scar formation.