The Expression And Ubiquitin-dependent Degradation Of SnoN Protein In Hypertrophic Scar Fibroblasts

Background Hypertrophic scars are the inevitable outcome of wound healing when theskin tissues suffer from depth damage. It’s usually induced by trauma, burn, surgery, and so on.The excessive proliferation of fibroblasts and extracellular matrix excessive synthesis anddeposition are the pathognomonic feature of hypertrophic scar. Transforming growth factor β1(TGF-β1) plays an important role in this pathological process. Signaling by TGF-β1is mainlymediated by the Smads proteins and the signal pathway subjects to various regulations. SnoN isa member of the Ski family of protein. The most important function of SnoN is to negativelyregulate TGF-β1signaling by interacting with the Smads proteins. So, the expressive level ofSnoN has a significant influence on the biological effects and is regulated byubiquitin-dependent pathway. The ubiquitin-proteasome pathway is an important t pathway forthe targeted degradation of proteins. This pathway plays a key role in a wide range of thephenomenon of life, including signal transduction, cell-cycle progression, replication andexpression of genetic information, and so forth. Ubiquitin proteasome pathway is composed ofubiquitin, a sequence of four enzymes and26S proteasomes. Recent studies have shown that thereduction of SnoN protein caused by ubiquitin-proteasome degradation is involved in theprogression of liver fibrosis, renal fibrosis and lung fibrosis. The mechanism is maybe thatdownregulation of SnoN enhancing the TGF-β1signal transduction. However, little is knownabout the expression and degradation of SnoN in hypertrophic scars fibroblasts.Objective To investigate the expression and ubiquitin-dependent degradation of SnoN inhypertrophic scars fibroblasts.Methods (1) Hypertrophic scar tissues from burn patients were collected and normalskin from the same patient which is the residual after the plastic surgery. Hypertrophic scar andnormal skin fibroblasts were cultured in vitro, and the locations of SnoN were observed byimmunofluorescence.(2) The cell lysates of hypertrophic scar fibroblasts and normal skin fibroblasts were collected and the expression of SnoN protein and mRNA were dectected byWestern Blot and RT-PCR.(3) Hypertrophic scar fibroblasts and normal fibroblasts were treatedwith10ng/ml TGF-β1for30min,1h,2h,6h, and the expression of SnoN protein and mRNAwere measured by Western Blot and RT-PCR.(4) The cell lysates of hypertrophic scar fibroblastsand normal skin fibroblasts were mixed with the ubiquitination reaction buffer, incubated for0-12h at37°C, and the reaction mixtures were subjected to Western Blot to detect thedegradation of SnoN protein using with anti-SnoN antibody.(5) Hypertrophic scar fibroblastswere performed for12h, with or without proteasome inhibitor (5μM MG132) to investigate theinhibition of degradation for SnoN.Results (1) In hypertrophic scar fibroblasts and normal skin fibroblasts, SnoN proteinswere expressed in the nucleus and cytoplasm, and were predominantly located in the cytoplasm.(2) SnoN protein expression in hypertrophic scar fibroblasts was lower than that in normal skinfibroblasts, however, there was no significant difference of SnoN mRNA expression betweenhypertrophic scar fibroblasts and normal skin fibroblasts.(3) With the TGF-β1stimulation, theexpression of SnoN mRNA and protein were increased in a time-dependent fashion in normalskin fibroblasts, while in hypertrophic scar fibroblasts, SnoN mRNA expression was increased ina time-dependent fashion, but SnoN protein expression was decreased in a time-dependentfashion.(4) Degradation of SnoN protein was increased in hypertrophic scar fibroblasts, whilethere was no significant reduction of SnoN protein in normal skin fibroblasts.(5) Thedegradation of SnoN protein was blocked by the proteasome inhibitor MG132.Conclusion Our results suggest that the reduction of SnoN protein in hypertrophic scarfibroblasts, which weakens its negative feedback effect and upregulates TGF-β1signaling, mayplay an important role in the progression of hypertrophic scarring. The mechanism of reductionof SnoN protein may be related to its ubiquitin-dependent degradation in hypertrophic scarfibroblasts.