|Year : 2013 | Volume
| Issue : 2 | Page : 46-50
The expression pattern of antiapoptotic protein c-FLIP in psoriatic epidermis
Adel A Imam, Al Hasan M El-Hefnawy, Shereen B El-Sayed, Sarah H Ibrahim
Department of Dermatology and Venereology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||26-Sep-2013|
|Date of Acceptance||26-Oct-2013|
|Date of Web Publication||31-Dec-2013|
Al Hasan M El-Hefnawy
Department of Dermatology and Venereology, Faculty of Medicine, Ain Shams University, Cairo
Source of Support: None, Conflict of Interest: None
Psoriasis is a chronic, relapsing, inflammatory, and hyperproliferative disease. The pathological changes affecting skin in psoriasis are mainly driven by abnormal differentiation secondary to activation of T cells. Keratinocytes may undergo apoptosis by loss of cell-cell contact after crosslinking of the FAS (CD95) molecule. The epidermal hyperplasia in psoriasis has been implicated to be a result of suppression of apoptosis. A key inhibitor of death receptor signaling is cellular FLICE inhibitory protein (c-FLIP), which interacts with FAS-associated death domain protein (FADD) and procaspase-8 preventing it from binding to caspase-8, thereby inhibiting the initiation of the apoptotic cascade.
Aim of work
This study was designed to investigate the expression pattern of antiapoptotic molecule c-FLIP in psoriasis vulgaris patients.
Materials and methods
Twenty psoriasis vulgaris patients were enrolled in this study. All patients were subjected to assessment of the degree of severity using PASI score. A 2-mm punch biopsy was taken from each patient from lesional and nonlesional areas for evaluation of c-FLIP mRNA gene expression using RT-PCR.
We found that there was a direct significant relationship between psoriasis and c-FLIP protein levels measured. The study also showed that the discrimination of the degree of severity of psoriasis using psoriatic skin c-FLIP mRNA level can be reliable, with 100% sensitivity, 81.2% specificity, and 85% accuracy.
Increased levels of c-FLIP might induce upregulation of keratinocyte proliferation and downregulation of the apoptotic signaling pathway in psoriatic epidermis. c-FLIP might be a potential new target for preventing keratinocyte proliferation in psoriasis in the future.
Keywords: cFLIP -apoptosis-proliferation-psoriasis.
|How to cite this article:|
Imam AA, El-Hefnawy AH, El-Sayed SB, Ibrahim SH. The expression pattern of antiapoptotic protein c-FLIP in psoriatic epidermis. Egypt J Dermatol Venerol 2013;33:46-50
|How to cite this URL:|
Imam AA, El-Hefnawy AH, El-Sayed SB, Ibrahim SH. The expression pattern of antiapoptotic protein c-FLIP in psoriatic epidermis. Egypt J Dermatol Venerol [serial online] 2013 [cited 2023 Jan 30];33:46-50. Available from: http://www.ejdv.eg.net/text.asp?2013/33/2/46/123929
| Introduction|| |
Psoriasis is a chronic, relapsing, inflammatory, and hyperproliferative skin disease  . Our understanding of the pathogenic events leading to psoriasis has improved significantly. The pathological changes affecting skin in psoriasis are mainly driven by abnormal differentiation secondary to activation of T cells or antigen-presenting cells, which in turn release various chemokines and cytokines that signal keratinocytes to hyperproliferate  .
Apoptosis or programmed cell death is critical for tissue homeostasis. Normally, in healthy skin the proliferation of cells in the basal cell layer is balanced and regulated by keratinocytes in the superficial layer of epidermis through the process of apoptosis  . Keratinocytes may undergo apoptosis by loss of cell-cell contact after crosslinking of the FAS (CD95) molecule  . The epidermal hyperplasia characteristic of psoriasis has been implicated to be a result of epidermal expression of apoptosis-related molecules leading to suppression of the apoptotic process  . A key inhibitor of death receptor signaling is cellular FLICE inhibitory protein (c-FLIP), which interacts with FAS-associated death domain protein (FADD) and procaspase-8, thereby inhibiting the initiation of the apoptotic cascade  .
The antiapoptotic c-FLIP protein
It is abundantly and constitutively expressed in a wide array of normal cell types  . The expression of c-FLIP has been proven to be one of the major determinants of resistance to death ligands such as FasL and TNF-related apoptosis inducing ligand (TRAIL)  . This protein contains two death effector domains (DEDs) that are highly homologous to the N-terminus of caspase-8 but lacks the enzymatic C-terminal portion of caspase 8. Many studies have shown that c-FLIP protein can be recruited into the DISC, where it binds to either FADD or caspase-8 through DED-DED ( = N-terminal DED interactions), resulting in inhibition of apoptosis  .
| Materials and methods|| |
This study was conducted on 20 psoriasis vulgaris patients attending the outpatient clinic of Ain Shams University Hospital. Age range was between 22 and 67 years, and the duration of the disease ranged from 1 week to 30 years. Disease severity was evaluated by the Psoriasis Area and Severity Index (PASI). Clinical information and collection of skin biopsies were obtained after taking written informed consent from all participants.
Skin biopsies (punch biopsy of 2 mm) were collected from each patient; two biopsies were obtained: the first was from involved psoriatic skin, whereas the second was from uninvolved skin at least 1 cm from the edge of the lesion, which served as the control group. Evaluation of c-FLIP mRNA gene expression using RT-PCR was performed by subjecting all tissue samples to the following: (a) RNA extraction and (b) amplification and quantification of c-FLIP mRNA. RNA was extracted from the PBMCs using MagNA Pure Compact Nucleic Acid Isolation Kit I (Cat. No. 03730964001) supplied by Roche, Mannheim (Germany). Amplification using RT-PCR was performed by Light Cycler-RNA Amplification Kit SYBR Green I (Cat. No. 2015137) (Roche). The amplicon was detected by measurement of the SYBR Green I fluorescence signal. Therefore, during PCR the increase in SYBR Green I fluorescence is directly proportional to the amount of double-stranded DNA generated.
Primer for c-FLIP and β-actin were supplied by Gulf-Tech, Portland, Oregon, USA (see [Table 1]).
The cycling profile used was as follows:
Denaturation at 95°C, annealing at 55°C, and extension at 72°C for 24 cycles.
It was performed by making a series of samples each with a different concentration using synthetic β-actin mRNA. A plot was constructed for the input copy number of the standards in relationship to the fluorescence of the product.
In this study, the results were expressed as a ratio of c-FLIP mRNA/β-actin mRNA.
β-actin amplification was performed as an internal control for each sample and the results were expressed as a ratio of c-FLIP mRNA/β-actin mRNA.
Setting of P-value (level of significance)
P -value more than 0.05: nonsignificant. P-value less than 0.05: significant (S). P-value equal to 0.01: highly significant.
To evaluate whether discrimination of the degree of severity of psoriasis using psoriatic skin c-FLIP mRNA level can be reliable or not, a ROC curve was used.
| Results|| |
Patients' age ranged between 20 and 67 years (mean ± SD = 44.1 ± 15.2). Sex distribution was 90% male patients (n = 18) and 10% female patients (n = 2). Of the 20 patients, 30% (n = 6) had positive family history of psoriasis and the remaining 70% (n = 14) had negative family history. Age at onset of psoriasis among patients ranged from 12 to 64 years (mean ± SD = 38.2 ± 17.3), with 40% (n = 8) early onset and 35% (n = 7) late onset. Disease duration ranged from 1 week to 30 years (mean ± SD = 6 ± 8.0). Assessment of the severity of psoriasis was made using PASI score, which ranged from 0.6 to 23.8 (mean ± SD = 7.1 ± 6.2); 80% (n = 16) had mild psoriasis, 15% (n = 3) had moderate psoriasis, and 5% (n = l) had severe psoriasis. The results were interpreted directly after completing the PCR. The level of c-FLIP in nonlesional skin ranged from 0.29 to 0.43 (mean ± SD = 0.35 ± 0.04), whereas in psoriatic skin ranged from 0.58 to 0.85 (mean ± SD = 0.72 ± 0.07) [Table 2].
There was a statistically significant variance between the expression level of c-FLIP mRNA in both specimens, as its expression in lesional biopsies was almost double than that in the nonlesional ones. There were interindividual differences between patients in many parameters. We correlated between age of the patient and age at onset, disease duration, severity of psoriasis (according to PASI score), and c-FLIP levels in both nonlesional and lesional areas.
Comparison of sex, family history, preceding infection, and associated autoimmune diseases with respect to c-FLIP levels showed that family history was the only significant factor (P < 0.05). The level of c-FLIP was higher in patients with negative family history (mean ± SD = 0.36% ± 0.04) than its level in patients with positive family history (mean ± SD = 0.32% ± 0.01).
In conclusion, putting all these parameters together revealed that the severity of psoriasis was the only significant factor (P<0.01) affecting the levels of c-FLIP and the relationship was directly related. The severity of disease with respect to c-FLIP was significant in nonlesional skin (P = 0.006), whereas the level was highly significant in lesional skin (P = 0.003). The statistical analysis not only showed a direct relationship between severity and c-FLIP levels, but also showed that c-FLIP levels in lesional biopsies were significantly higher than that in the nonlesional ones.
ROC curve to discriminate moderate/severe cases from mild cases using psoriatic skin c-FLIP mRNA
A high statistical significance (P < 0.01) was detected between the severity of psoriasis and c-FLIP levels.
To evaluate the degree of severity of psoriasis using psoriatic skin c-FLIP mRNA level, a ROC curve was used, which showed 100% sensitivity, 81.2% specificity, and 85% accuracy [Table 3].
| Discussion|| |
Dysregulation of c-FLIP expression has been shown to be associated with various diseases such as cancer and autoimmune diseases [11-13]. c-FLIP was upregulated to levels sufficient to prevent death receptor-induced apoptosis in many human tumor cells, including colorectal cancer  , melanoma  , and ovarian carcinoma  . Therefore, our results implicate c-FLIP expression as an important mechanism protecting keratinocytes from apoptotic elimination. Hence, it has been reported that decreased c-FLIP levels enhance the sensitivity of tumor cells not only to Fas but also to TRAIL-mediated apoptosis  . These data render c-FLIP an attractive candidate to predict the effectiveness of future immune therapies in colon cancer  . Therefore, c-FLIP might be a potential new target for preventing keratinocyte proliferation in psoriasis in the future.
Upregulation of the cell survival pathways and suppression of apoptosis are implicated in the development of psoriasis  . Several apoptotic inhibitors have been shown to be elevated in neoplasms and in some inflammatory conditions such as psoriasis  . Psoriatic keratinocytes have an increased capacity to resist apoptosis. Few studies have indicated that apoptosis may be a relevant mechanism in plaque psoriasis  . Alterations in the sensitivity and resistance to death receptor-mediated apoptosis have been reported to be involved in autoimmune diseases; data obtained from cells overexpressing c-FLIP and from mice deficient in c-FLIP clearly support an antiapoptotic function  . In many in-vitro studies, FLIP proteins have been found to protect cells against apoptosis induced by several death receptors due to their high structural homology with procaspase-8  . Overexpression of c-FLIP in lesional psoriatic skin might contribute to abnormal keratinocyte proliferation due to a functional decrease in the apoptotic pathway  . On the background of previous reports, we can see that there is a relationship between inhibitors of apoptosis, for example, c-FLIP protein, and psoriasis.
In this study, we analyzed the levels of mRNA of the apoptotic inhibitor c-FLIP in the epidermis of both nonlesional and lesional samples using RT-PCR, nonlesional samples serving as controls.
Correlations were made to determine different parameters in psoriasis that affect the level of c-FLIP protein and this included sex, age, family history, age at onset of psoriasis, duration of the disease, and preceding infection. All these parameters had no effect on the level of c-FLIP protein, and the relationship was nonsignificant (P > 0.05).
Little is known about the expression of c-FLIP protein in psoriatic epidermis and whether or not its level has an impact on the severity of the disease. It was found that the level of c-FLIP protein mRNA in lesional skin was nearly double than that of the nonlesional one in each patient. These results indicated that c-FLIP protein plays an important role in the development of psoriasis. Further assessment was performed to evaluate the relationship between the PASI score and c-FLIP protein levels and to determine the role of increased c-FLIP on the severity of psoriasis. There was a significant positive correlation between c-FLIP expression in lesional epidermis and the PASI scores. This result indicated that increased levels of c-FLIP in psoriatic skin were highly related to the clinical severity of psoriasis. Further large-scale studies are recommended for additional assessment of the role of c-FLIP protein in the development of psoriasis and its severity.
Defects in apoptosis contribute in many diseases, ranging from cancer and autoimmunity to degenerative disorders. In mammalian cells, there are two pathways to apoptosis - the 'extrinsic' pathway and the 'intrinsic' pathway - each activating different initiator caspases, but they converge at the level of effector caspases. Inhibitors of the apoptosis signaling pathway exist and play a major role in the apoptotic process. A key apoptosis regulatory protein of the Fas death pathway is c-FLIP. FLIP has been shown to prevent the binding of procaspase-8 to FADD or interfering with the autocatalytic activation of caspase-8 at the death-inducing signaling complex, leading to failure of the initiation of the caspase cascade of the death receptor-mediated extrinsic apoptotic pathway. However, there was a negative correlation between c-FLIP expression and AI (Spearman's P = −0.41, P < 0.05).
c-FLIP mRNA expression was detected in all clinically involved psoriasis biopsies using RT-PCR. Although interindividual differences in c-FLIP mRNA expression were observed, the level of c-FLIP mRNA expression in lesional psoriatic skin was higher compared with that in nonlesional psoriatic skin. Furthermore, the elevation of the mean c-FLIP mRNA expression in lesional psoriatic skin compared with nonlesional psoriatic skin was statistically significant (3.8-fold and 3.6-fold, P < 0.01, mean ± SEM).
| Conclusion|| |
Our results implicate c-FLIP expression as an important mechanism protecting keratinocytes from apoptotic elimination. Therefore, c-FLIP might be a potential new target for preventing keratinocyte proliferation in psoriasis in the future. This suggested that the increased levels of c-FLIP might induce upregulation of keratinocyte proliferation and downregulation of the apoptotic signaling pathway in psoriatic epidermis. Several lines of evidence strengthen this finding. Several antipsoriatic agents also inhibit keratinocyte proliferation and increase keratinocyte apoptosis. These include propylthiouracil, steroids, methotrexate, cyclosporine A, and calcipotriol. Further investigation is required to identify whether c-FLIP takes part in promoting the keratinocyte apoptosis induced by these antipsoriatic agents in psoriasis and whether it could be used as a parameter for the success of these treatments. These results suggest that antiapoptotic c-FLIP protein may play a prominent role in psoriasis epidermal hyperplasia.
| Acknowledgements|| |
Conflicts of interest
| References|| |
|1.||Barker J. Skin diseases with high public health impact (psoriasis). Eur J Dermatol 2007; 17:563-564. |
|2.||Gudjonsson JE, Johnston A, Sigmundsdottýr H, Valdimarsson H. Immunopathogenic mechanisms in psoriasis. Clin Exp Immunol 2004; 135:1. |
|3.||RR Polakowska, Piacentini M, Bartlett R, et al. Apoptosis in human skin development: morphogenesis, periderm and stem cells. Dev Dyn 1994; 199:176-188. |
|4.||Krueger A, Baumann S, Krammer PH, et al. FLICE inhibitory proteins: regulators of death receptor-mediated apoptosis. Mol Cell Biol 2001; 21:8247-8254. |
|5.||Takahashi H, Manabe A, Ishida-Yamamoto A, et al. Aberrant expression of apoptosis-related molecules in psoriatic epidermis. J Dermatol Sci 2002; 28:187-195 |
|6.||Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP. Nature 1997; 388:190-195. |
|7.||Marconi A, Atzei P, Panza C, et al. FLICE/caspase-8 activation triggers anoikis induced by (beta) 1-integrin blockade in human keratinocytes. J Cell Sci 2004; 117:5815-5823. |
|8.||Timothy R Wilson, McLaughlin KM, McEwan M, et al. c-FLIP: a key regulator of colorectal cancer cell death. Cancer Res 2007; 67:5754-5762. |
|9.||Budd RC, Yeh WC, Tschopp J. c-FLIP regulation of lymphocyte activation and development. Nat Rev Immunol 2006; 6:196-204. |
|10.||Braun K, Hirsch B, Al-Yacoub N, et al. Resistance of cutaneous anaplastic large-cell lymphoma cells to apoptosis by death ligands is enhanced by CD30-mediated over expression of c-FLIP. J Invest Dermatol 2010; 130:826-840. |
|11.||Xu L, Zhang L, Yi Y, et al. Human lupus T cells resist inactivation and escape death by upregulating COX-2. Nat Med 2004; 10: 411-415. |
|12.||Mathas S, Lietz A, Anagnostopoulos I, et al. c-FLIP mediates resistance of Hodgkin/Reed-Sternberg cells to death receptor-induced apoptosis. J Exp Med 2004; 199:1041-1052. |
|13.||Fredriksson T, Pettersson U Severe psoriasis - oral therapy with a new retinoid. Dermatologica 1978; 157:238-244. |
|14.||Wilson TR, McLaughlin KM, McEwan M, et al. c-FLIP: a key regulator of colorectal cancer cell death. Cancer Res 2007; 67: 5754-5762. |
|15.||Abedini MR, Qiu Q, Yan X, et al. Possible role of FLICE-like inhibitory protein (FLIP) in chemoresistant ovarian cancer cells in vitro. Oncogene 2004; 23: 6997-7004. |
|16.||Wang Q, Wang X, Zhou Y, et al. PKCdelta-mediated regulation of FLIP expression in human colon cancer cells. Int J Cancer 2006; 118:326-334. |
|17.||Galligan L, Longley DB, McEwan M, et al. Chemotherapy and TRAIL-mediated colon cancer cell death: the roles of p53, TRAIL receptors, and c-FLIP. Mol Cancer Ther 2005; 4:2026-2036. |
|18.||Bowen AR, AN Hanks, KJ Murphy, et al. Proliferation, apoptosis and survivin expression in keratinocytic neoplasms and hyperplasias. Am J Dermatopathol 2004; 26:177-181. |
|19.||Weatherhead S 2010 The role of apoptosis in UVB-induced clearance of psoriasis Faculty of Medical Sciences, Institute of Cellular Medicine, Newcastle, London University, England. |
|20.||CEM Griffiths, BE Strober, et al. Comparison of Ustekinumab and Etanercept for moderate-to-severe psoriasis. N Engl J Med 2010; 362:2. |
|21.||Yang J, Yan LI, Ye-Qiang LIU, et al. Expression of anti-apoptotic protein c-FLIP is up-regulated in psoriasis epidermis. Eur J Dermatol 2009; 19:29-33. |
[Table 1], [Table 2], [Table 3]