Effects And Possible Mechanism Of EIF6 On Mechanotransduction In Murine Dermal Fibroblasts

Background:Pathological scarring,including hypertrophic and keloid scarring,is a common and clinically significant complication after severe burn injuries.The matrix proteins secreted by hypertrophic scar fibroblasts differ from those secreted by normal skin cells.The extra cellular matrix(ECM)in hypertrophic scars never achieves the flexibility or strength of the original tissue,often leading to physical dysfunction and psychological stress.At present,the mechanisms underlying hypertrophic scar formation are not fully understood.Mechanical loading profoundly influences the composition and structure of the ECM.Many in vivo studies have shown that keloid and hypertrophic scars have few cellular abnormalities and often occur at sites that are constantly or frequently subjected to external mechanical skin stretching.Tension during healing appears to provoke an excessive or deranged response,resulting in fibrogenesis and increased deposition of collagen.This response suggests that mechanosensory pathways may be responsible for mechanical force-mediated fibrogenesis.The structure and function of dermal fibroblasts,the major cell type involved in wound repair,are affected by mechanical loading.Local mechanical forces within an open wound subject dermal fibroblasts to internal forces,either by extending their membranes or by rearranging their actin cytoskeletons.In this situation,characterized by increased ?-smooth muscle actin(?-SMA)expression,collagen synthesis and contraction,an abnormally large number of dermal fibroblasts differentiate into myofibroblasts.Transforming growth factor-?(TGF-?)is one of the most important molecules contributing to the activation and maintenance of myofibroblasts.In homeostatic conditions,TGF-? is sequestered in the ECM as part of the large latent complex(LLC),which includes latency-associated peptides(LAPs)and latent TGF-?-binding proteins(LTBPs).Upon injury,the binding of integrins to LTBPs,together with increased mechanical forces,can cause the release of TGF-? from the LLC,allowing it to interact with two type I and two type II receptors and subsequently with phosphorylated Smad proteins,resulting in the formation of a hypertrophic scar.Cyclic mechanical strain results in a significant increase in active TGF-?1 levels,leading to a p-Smad2-mediated increase in the transcription of downstream regulatory factors.These observations suggest that the activity of TGF-?1 plays an important role in the formation of hypertrophic scars under external mechanical loading,and that the identification of a TGF-?1 inhibitory factor will be a key step in the research for treatments to control hypertrophic scarring.Recently,eukaryotic initiation factor 6(eIF6),also known as protein p27BBP(beta 4 binding protein),has been identified as a regulator of TGF-?1 expression and myofibroblast differentiation.eIF6 is also involved in the development of hypertrophic scars,and is therefore a potential target for the control of hypertrophic scarring.Our earlier study showed that eIF6 regulates TGF-?1 expression at the transcriptional level by facilitating occupancy of the TGF-?1 promoter by Sp1 rather than H2 A.Z.In vivo studies also show that a heterozygous eIF6+/-mouse exhibits enhanced TGF-?1 production after skin injury,coupled with increased numbers of ?-smooth muscle actin(?-SMA)+ myofibroblasts.We hypothesized that eIF6 plays a role in maintaining fibroblast homeostasis under external mechanical loading through TGF-? pathway regulation.The eIF6 protein has five quasi-identical ?/? subdomains that link integrin a6?4 to the intermediate filament cytoskeleton and form a multivalent laminin receptor.These findings suggest to us that eIF6 may be a mechano-sensitive protein,essential for cytoskeletal integrity.In this study,we examined the expression of eIF6 and components of TGF-?/Smad signaling pathway in cell stretch and dermal stretch models to determine their effects on murine dermal fibroblasts.Motheds:In this study,the expressed eIF6 and components of TGF-?/Smad signaling pathway and their effects on murine dermal fibroblasts have been examined in a cell stretch model as well as in a mice dermal stretch model.Primary murine dermal fibroblasts were isolated from the skin of wild type(eIF6+/+)and eIF6 heterozygous(eIF6 deficiency,eIF6+/-)newborn C57BL/6 murine,and were cultured on Bioflex? six holes plates,combined with or without LY2109761(a dual inhibitor for both TGF-? receptor type I and type II),or dimethyl sulfoxide(DMSO).The dermal fibroblasts were subjected to cyclic tension at a frequency of 0.1 Hz(6 cycles/min)with a maximal increase in surface area of 10 % for 4h,8h,16 h and 24 h.Partial-thickness wounds were subjected to external mechanical stretch for 5 days,with or without LY2109761,or DMSO injection.A stable animal model were established by an novel mouse partial-thickness wound modeling tools.The murine partial-thickness wound were dissected and immediately fixed in 4% paraformaldehyde in 0.1 M PBS at p H 7.4 for over 48 h,and embedded with paraffin.Successive transverse paraffin sections were cut at ~4um thickness and underwent HE staining as well as Masson's trichrome staining.Immunohistochemistry and Immunofluorescence analyses was performed on formalin-fixed,paraffin-embedded tissue as well as dermal fibroblasts to test the expression of eIF6,Proliferating cell nuclear antigen(PCNA)and COL1A1.Western blotting was performed to detect the protein expression of eIF6,TGF-?1,PCNA,COL1A1,Smad2,p-Smad2 and p-Smad7.Quantitative RT-PCR was performed to detect the m RNA expression of eIF6,PCNA and COL1A1.All data were expressed as mean ± SD and statistically compared by Dunnett's multiple-comparison tests as well as Paired samples T-test(two-tailed),and post-hoc tests were undertaken using the Graph Pad Prism software.Significance was indicated as *P < 0.05 and **P < 0.01.Results:External mechanical stretch significantly increased the COL1A1,TGF-?1 and eIF6 expression as well as proliferation of normal dermal fibroblasts both in vitro and in vivo.In the in vitro study,the COL1A1 expression of eIF6 deficiency(eIF6+/-)dermal fibroblasts cells significantly increased under mechanical stretch compared to normal dermal fibroblasts,with a significantly improvement of TGF-?/Smad signal.Under the condition of a 5-days external mechanical stretch of eIF6 heterozygous mice it was proved that eIF6 deficiency significantly increased the granulation tissue formation in partial thickness wounds.Under this condition,an inhibition of TGF?RI/II by LY2109761,a dual inhibitor of both TGF-? receptor type I and type II,showed that LY2109761 could significantly decrease the COL1A1 protein expression in eIF6+/-murine dermal fibroblasts in the cell stretch model,and attenuate the granulation tissue formation in the partial thickness wound of eIF6+/-mice.Conclusion:eIF6 might be involved in external mechanical force-mediated dermal fibroblast function through,at least partly,the TGF-? pathway.