Mechanistic Study Of TGFβ1/Smad Signaling Pathway In Actinic Keratosis

Part I:Analysis of clinical and pathological features of actinic keratosisObjective:The incidence of actinic keratosis (AK) increases year by year, and AK could transform into squamous cell carcinoma (SCC) further, which is serious harmful to people’s lives and health. In order to improve clinical diagnosis, prevention and treatment level and to minimize morbidity and mortality, the clinical and pathological features of AK was investigated to complete epidemiological data of AK in plateau region.Methods:Total histopathological confirmed52AKs, which with complete clinical data, were studied retrospectively from department of dermatology of First Affiliated Hospital of Kunming Medical University during2011to2012, and the pathological features were also observed and analyzed carefully. The data were collected and analyzed using SPSS17.0software.Results:There were52cases of AK diagnosed pathologically between2011and2012. Confirmed AK accounted to0.49%and0.85%of annual pathological examination number, and for annual outpatient ratio was0.18%o and0.34%o respectively. The annal outpatient ratio of2012was increased significantly comparing with2011(x2=5.02, p=0.03);52cases of AK included18males and34females, male:female was1:1.89; The age of onset was45to82years, among them<50years was4cases (7.7%),50to59years was11cases (21.2%),60to69years was17cases (32.7%),70to79years was17cases (32.7%),≥80years was3cases (5.8%), the average age of onset was64.98±9.78,the difference of average age of onset beteween males (62.44±10.76) and femals (66.32±9.11) was not significant (t=-1.372, p=0.176); The main occupation of patients was outdoor workers, such as farmers and construction workers, reached to34cases (65.3%), the diseased parts, age of onset and duration among different occupations were not significantly different (p>0.05); There were27cases (42.3%) of Type III skin according to sun-reactive skin typing, and25cases (57.7%) of Type IV skin, the age of onset and duration between different skin types were not significant different (p>0.05); The main cilinical feature of AK lesions was red-brown, dark-brown pimples with scarly, which accounting to46cases (88.5%), there was one case merged with ulcer and erosion, only the black papules was5cases (9.6%), and cutaneous horn-like was one case (1.9%), all lesions located in the sun-exposed areas of skin, there were25cased in cheek (48.1%),23cases in temporal (44.2%),13cases in nose (25%),11cases in frontal (21.2%), and one in ear (1.9%), one in neck (1.9%), one in hand (1.9%); AK lesions were mainly single (71.2%) in one patient, there were6patients had multi-lesions and the maximum reached21lesions in one patient; The mean lesion diameter was17.87±9.92mm, diameter of lesions was not sifnificantly different between different anatomical site (p>0.05); The mean disease course was44.04±53.09months, male mean disease duration was50.39±29.82months, and female was40.68±62.16months, the average duration of disease between men and women had no significant difference (p>0.05); The dominant pathological type was hypertrophic (51.9%), followed was atrophy (21.2%) and pigmented (15.4%), while acantholytic type (7.7%) and Bowenoid type (3.8%) are relatively rare, and there were2patients combined with SCC; the clinical and pathological diagnosis compliance rate was51.9%among52patients, which was misdiagnosed as seborrheic keratosis13cases, basal cell carcinoma2cases, nevi4cases, common warts3cases, cutaneous horn2cases, and1case of keratoacanthoma.Conclusion:1. The proportion of diagnosed AK in annual outpatient visits represented an upward trend, which should arouse the whole society’s attention;2. AK more often occured in sun-exposed areas of women over the age of50, and main occupation of AK patients was outdoor workers, which demonstrated that close relationship of AK and ultraviolet radiation;3. The clinical features of AK was dominantly represented as red-brown, dark-brown pimples with scarly, mean disease duration was3.6years, and the lesion was single with the average diameter over than1cm;4. The main pathological type was hypertrophic, the low clinical and pathological diagnosis compliance rate needs for the extending of pathological examination, to avoid delaying the disease, which leading to serious consequences;5. The transformation rate of AK to SCC was high, which should be early diagnosis and treatment.Part II:Mechanistic study of TGFβ1/Smads signaling pathway in actinic keratosisObjective:The high incidence of actinic keratosis (AK), the precursor of squamous cell carcinoma (SCC), is induced by prolonged exposure to ultraviolet (UV) radiation. UV radiation could also promote transformation of AK to SCC, which is serious harmful to people’s lives and health. Transforming growth factor beta1(TGF(31)/Smad signaling pathway acts as a tumor suppressor in early stages, but promotes tumor invasion in later stages. However, the mechanisms of this pathway in AK have not been studied before. In this study, the mechanisms of TGFβ1/Smads signaling pathway in AK was investigated to clarify the pathogenesis of AK.Methods:AK explants and primary cells were cultured and divided into3groups, Group I is control group, Group II is TGFβ1addition group, and Group III is SB431542addition group. After48h treatment, the tissues and cells were collected respectively for:A. the proliferation and apoptosis analysis with flow cytometry, Ki-67and TUNEL staining; B. the detection of TβRII and Smad2expression changes with real-time PCR and Western blot in AK explants to explore the feasibility of cultured tissue for pathway interference; C. The detection of expression changes of p53, Fas and FasL in AK explants with real-time PCR and Western blot. Results:A. After12d culture, the keratinocytes of AK showed a typical cobblestone change. The cultured tissues grew well, no significant apoptosis and death was observed. B. After TGFβ1administration, the number of keratinocytes (99.80±6.06), and the percentage of Ki-67was decreased (0.42±0.04) in addition group, the percentage of Ki-67was also decreased in AK tissues (3.90±0.24)(p<0.05). C. Comparing to control, mRNA of Smad2(0.01791±0.00080), the phosphorylated level of TpRI (0.1858±0.0107) and Smad2(0.6112±0.0391) was increased after TGFβ1addition, and the phosphorylated TβRI (0.0644±0.0053) was decreased after SB431542treatment (p<0.05). D. Compared with control, the mRNA (0.00178±0.00010) and protein (0.1620±0.0155) of p53, and the protein (0.4004±0.0267) of Fas was increased (p<0.05).Conclusion:1. ex vivo culture of skin tissue can be used as the signaling pathway interference model.2. TGFβ1/Smad signaling pathway plays an inhibition role in AK.3. TGFβ1/Smad signaling pathway exerts inhibition effect in AK through induction of p53and Fas.Part III:The effects of UV radiation on TGFβ1/Smad signalling pathway in AK Objective:UV radiation could promote transformation of AK to SCC, which is serious harmful to people’s lives and health. TGFβ1/Smad pathway acts as a tumor suppressor in AK. However, the influence of UV on TGFβ1/Smad signaling pathway have not been studied before. In this study, the effects of UV radiation on TGFβ1/Smad in AK were investigated to clarify the machnisms of progression of AK to SCC.Methods:The human normal skin and AK tissues were cultured and divided into four groups:OJ/cm2group(control group),5J/cm2group,10J/cm2group, and20J/cm2group. The tissues were respectively radiated on four consecutive days.24hours after radiation, tissues were collected for real time PCR and immunohistochemistry detection of TGFβ1, TβRI, TβRII, Smad2, Smad3, Smad4and Smad7.Results:A. Comparing to control, the percentage of positive Ki-67cells decreased in the20J/cm2group of normal skin (3.74±1.16)(p<0.05), but was not significantly changed in AK. Compared with control, the TUNEL-positive cells in20J/cm2group of normal skin (39.34±8.73) had increased (P<0.05), and so did the20J/cm2group of AK (25.76±5.99)(P<0.05).B.After radiation,comparing to control,the mRNA and protein level of TGFβ1in the10(0.00821±0.00181;0.1041±0.0064) and20J/cm2(0.01040±0.00145;0.1257±0.0135) groups of normal skin and in the20J/cm2group (0.00504±0.00122;0.0871±0.0038) of AK was up-regulated. However, TβRII, the membrane receptor of TGFβ1, was down-regulated in the20J/cm2groups of both normal skin (0.00119±0.00019;0.0366±0.0052) and AK (0.00073±0.00029;0.0343±0.0043). The mRNA (0.00519±0.00119) and phosphorylated level of Smad2(0.0160±0.0036) was only reduced in the20J/cm2group of AK. In contrast, the inhibitor of TGFβ1/Smad pathway, Smad7, was increased in the20J/cm2groups of both AK (0.00055±0.00012;0.0096±0.0009) and normal skin (0.00028±0.00006;0.0093±0.0003)(P<0.05). The expression of TβRI, Smad3and Smad4had no significant changes (P>0.05).Conclusion:1. The suppression of TGFβ1/Smad pathway by UV radiation may contribute to the progression of AK to SCC.2.The suppression of UV radiation on TGFβ1/Smad signaling pathway may be associated with the induction of Smad7.