The etiology of psoriasis is complex and not well understood. T-cells are almost certainly involved in the initiation and maintenance of psoriatic lesions through elaboration of TH1 cytokines . A prominent role for the epidermal growth factor (EGF) receptor–ligand system in psoriatic hyperplasia is strongly suggested . Seven mammalian EGF receptor (EGFR) ligands have been identified. EGF, transforming growth factor-α, amphiregulin, and epigen bind only to the EGFR, whereas heparin-binding EGF-like growth factor, β-cellulin, and epiregulin also bind to hepatocyte receptor 4 .
Amphiregulin is a heparin-binding, heparin-inhibited member of the EGF family and is the predominant autocrine growth factor for cultured human keratinocytes . It is an important regulator of cellular growth in keratinocytes, carcinomas, and hyperproliferative epidermal disorders, including psoriasis. It is synthesized as a 252-amino acid precursor protein, which undergoes posttranslational modifications by N-linked glycosylation and proteolytic processing .
Although amphiregulin is one of the EGFR ligands involved in keratinocyte hyperproliferation, its role in psoriasis is more complex as it was found that amphiregulin has a specific functional role in enhancing transmigration of neutrophils that is not shared by other EGFR ligands, which are also overexpressed in psoriatic epidermis. Moreover, amphiregulin markedly alters E-cadherin expression, localization, and processing in the epidermis of amphiregulin-transgenic mice similar to that observed in the involved skin of individuals with psoriasis. Thus, amphiregulin acts as a dual activity protein, possessing EGFR-mediated mitogenic activity and proinflammatory activity that can stimulate both psoriatic epidermal hyperproliferation and psoriatic inflammation .
It is to be noted that loss of function of the epithelial barrier that accompanies alterations of epithelial junctional molecules, such as E-cadherin, P-cadherin, zona occludens 1, and occludin may explain the gut-related barrier disruption seen in inflammatory bowel disease associated with psoriasis [7,8].
Amphiregulin may also mediate critical events in the cutaneous wounding response, including reepithelization and epidermal barrier function [9,10]. These findings may provide an explanation for the role of amphiregulin in epidermal repair as well as the dysregulated expression and activity that contribute to the exaggerated isomorphic (koebner) response in psoriasis .
Johnston et al.  reported that high concentration of leptin may further induce local production of amphiregulin, thus explaining the relationship between obesity and psoriasis.
It is to be noted that amphiregulin binds to heparin and this binding disrupts the interaction of amphiregulin with the EGFR. Thus, administration of exogenous heparin or means of stimulating endogenous heparan sulfate synthesis might be of therapeutic value in psoriasis .
The aim of this study was to detect the pathogenic role of amphiregulin in psoriasis by comparing its level in psoriatic lesions with healthy control skin and by investigating whether its level would be altered after psoralen with ultraviolet A (PUVA) therapy or not.
Patients and methods
This study was conducted on patients suffering from psoriasis vulgaris (plaque type) who were treated at the phototherapy unit of Kasr El Aini Hospital's Dermatology Department, Cairo University.
The study included 15 patients with psoriasis (plaque type) and 15 controls of healthy individuals as volunteers matching the same age group. Each of the patient and control groups signed a written consent after explanation of the aim of the study and procedures involved.
All the patients were cases presenting at the outpatient clinic and had not received any topical treatment for 2 weeks and no systemic treatment for 1 year before start of PUVA sessions. Each patient was subjected to full medical history and examination, in addition to routine laboratory investigations. Exclusion criteria included pregnant cases, patients suffering from any other autoimmune diseases, and cases having altered liver or kidney function tests. The Psoriasis Area and Severity Index (PASI) score, which includes assessment of erythema, infiltration, desquamation, and extent of lesions , was calculated for each patient.
A 5 mm punch skin biopsy specimen was taken from each patient before starting photochemotherapy. Each biopsy was divided into two parts: one part was stained by hematoxylin and eosin for histopathological confirmation of the diagnosis and the second part was processed for amphiregulin detection by reverse transcriptase polymerase chain reaction (RT-PCR) and quantitation by a gel documentation system.
All the patients underwent PUVA therapy. We started the treatment with the minimal erythema dose of 0.25 J/cm2. The dose was increased to the next increment every other session according to the response, which varied from one case to another. A second 5 mm punch skin biopsy from the same site of the previous one was taken from all the patients after clinical cure (improvement in PASI score more than 90%) for amphiregulin detection by RT-PCR and quantitation by a gel documentation system. Furthermore, skin biopsy was taken from each control individual. All the biopsies were put in RNA extraction buffer and then kept frozen at −80°C for further use.
Psoriatic skin and control biopsies were homogenized at high speed using a small homogenizer until no visible tissue fragments remained. Then, centrifugation was performed at 10 000 rpm for 10 min and the supernatant was used for RNA extraction. Total RNA was extracted from the cells using an SV total RNA isolation system kit (Promega, Madison, Wisconsin, USA) according to the manufacturer's recommendations.
Reverse transcriptase polymerase chain reaction
RT-PCR experiments were conducted for detecting amphiregulin gene expression using specific primer sequences. The primers were prepared using the oligo-1000 DNA synthesizer from Beckman, California, USA. The primer sequences were amphiregulin sense 5′-TTGATACTCGGCTCAGGCC-3′ and antisense 5′-CTACTGTCAATCATGCTGTGA-3′. cDNA was generated with 5 μl (20 pmol) of oligo (dt) primer and 0.8 μl of superscript RNase H reverse transcriptase for 45 min at 37°C.
For PCR, 4 μl of complementary DNA was incubated with 30.5 μl of water, 4 μl of mgcl2 (25 mmol/l), 1 μl of dNTPs (10 Mm), 5 μl of 10X PCR buffer, 0.5 μl of (2.5u) Taq polymerase, and 2.5 μl of each primer containing 10 pmol. The reaction mixture was subjected to 39 cycles of PCR amplification as follows: denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 2 min.
The PCR product was detected using agarose gel (1.5%) electrophoresis, and the band appeared at 539 bp. The UV-illuminated gels were photographed and the densitometry was analyzed using a Gene Genius gel documentation system (Syngene, Cambridge, UK). The densitometry was performed at the Agricultural Scientific Research Center, Cairo, Egypt.
Data was coded and entered using the statistical package for social science version 15 (SPSS Inc., Chicago, Illinois, USA). Data were summarized using mean±standard deviation (SD) for quantitative variables and percentage for qualitative variables. Comparisons between groups were performed using Student's t-tests. Correlation was performed to test the linear relation between quantitative variables. A P value of less than and equal to 0.05 was considered statistically significant and P value of less than or equal to 0.001 was considered highly significant.
The study included 15 patients with plaque-type psoriasis (seven male and eight female patients). Their ages ranged from 14 to 63 years with a mean age of 35.13±16.1 years and 15 healthy age-matched controls (six male and nine female participants) were also included. The disease duration ranged between 9 months and 35 years with a mean of 7.78±8.7 years. The PASI score varied from 2.4 to 27 with a mean of 15.75±7.26.
By measuring the amphiregulin level, it was found that its mean level in patients before treatment (412.066±76.38) was significantly higher than that of controls (89.61±24.60) (P<0.001) (Table 1 and Fig. 1).
Moreover, the mean values of amphiregulin was statistically significantly decreased in patients after treatment (180.66±28.5) than those before treatment (412.066±76.38) (P<0.001) (Table 2, Fig. 2). No correlation was detected between the amphiregulin level before treatment and each of disease duration, PASI score, and age (r=0.136, P=0.629), (r=0.190, P=0.497), and (r=0.110, P=0.239), respectively.
Amphiregulin, one of the several EGFs, has been implicated in stimulating epidermal proliferation in both psoriasis  and squamous cell carcinoma [15,16].
In contrast to EGF, amphiregulin might contribute to the pathogenesis of psoriasis not only by its mitogenic effect on the keratinocytes but also by affecting the integrity of cell–cell junction through the reduction of E-cadherin, thus facilitating the neutrophil and lymphocyte migration into the psoriatic epidermis .
To evaluate the role of amphiregulin in psoriasis, this study assessed the values of amphiregulin in the lesional skin of 15 patients with psoriasis (seven male and eight female patients) both before and after treatment by PUVA and compared them with those of 15 healthy controls as volunteers (six male and nine female volunteers) matching with the same age group.
The mean amphiregulin level in patients before photochemotherapy was significantly higher (P=0.001) than those of controls. This result confirmed that of Cook et al. , who have demonstrated that involucrin promoter-driven expression of human amphiregulin in the suprabasal epidermis of transgenic mice results in an early-onset, severe skin phenotype that strongly resembles the cutaneous lesions of psoriasis. In addition, these results are in agreement with those obtained by Bhagavathula et al.  who demonstrated that intraperitoneal injection of a humanized antiamphiregulin monoclonal antibody into severe combined immunodeficient mice effectively reverses the epidermal hyperplasia present in human psoriatic lesional skin transplanted into the mice.
In contrast, Yoshida et al.  by studying the expression of amphiregulin in normal and psoriatic skin biopsies, found that amphiregulin was positive throughout the epidermis in almost all normal specimens. In contrast, it was generally negative in the basal layer and became only positive in the upper spinous layer of the psoriatic skin specimens. They also reported that amphiregulin did not promote the growth of adult-type keratinocytes in vitro. Hence, they concluded that amphiregulin might not be so important in the epidermal proliferation of psoriasis.
In addition, Takahashi et al.  by measuring the levels of various cytokines and growth factors in the sera of patients with psoriasis and by comparing them with those of healthy controls, found no significant difference in the level of amphiregulin between both groups.
To study the effect of photochemotherapy on amphiregulin, we compared the mean values of amphiregulin in patients before PUVA with those after treatment. A statistically significant decrease in the values of amphiregulin was detected after PUVA. To the best of our knowledge, the above results represent the first prospective study to evaluate the effect of photochemotherapy on the level of amphiregulin in lesional skin of patients with psoriasis.
There was a positive nonsignificant correlation between the amphiregulin level before treatment and both the PASI score and the disease duration. No significant correlation could be detected between the amphiregulin level and either the age or the sex of the patients.
Results of this study suggest that amphiregulin might play an important role in the pathogenesis of psoriasis. Hence, an anti-amphiregulin strategy (heparin, heparin-like compounds as heparan sulfate) may have therapeutic efficacy in psoriasis and the associated inflammatory bowel diseases. The significant decrease in the levels of amphiregulin after photochemotherapy may be considered as one of the mechanisms by which PUVA treats psoriasis.
Further studies in this area should include evaluation of both tissue and serum levels of amphiregulin with other therapeutic modalities of psoriasis. In addition, studying the effect of amphiregulin antagonists such as heparin, heparan sulfates, and glucosamine in the treatment of psoriasis may provide a promising safe and specific therapeutic modality of this chronic disease.
There is no conflict of interest to declare.
1. Austin LM, Ozawa M, Kikuchi T, Walters IB, Krueger JG. The majority of epidermal T cells in Psoriasis
vulgaris lesions can produce type 1 cytokines, interferon-gamma, interleukin-2 and tumor necrosis factor-alpha, defining TC1 (cytotoxic T lymphocyte) and TH1 effector populations: a type 1 differentiation bias is also measured in circulating blood T cells in psoriatic patients. J Invest Dermatol. 1999;113:752–759
2. Bahagavathula N, Nerusu KC, Fisher GJ, Liu G, Thakur BA, Gemmell L, et al. Amphiregulin
and epidermal hyperplasia: amphiregulin
is required to maintain the psoriatic phenotype of human skin grafts on severe combined immunodefficient mice. Am J Pathol. 2005;166:1009–1016
3. Harris RC, Chung E, Coffey RJ. EGF receptor ligands. Exp Cell Res. 2003;284:2–13
4. Cook PW, Brown JR, Cornell KA, Pittelkow MR. Suprabasal expression of human amphiregulin
in the epidermis of transgenic mice induces a severe early-onset, psoriasis
-like skin pathology: expression of amphiregulin
in the basal epidermis is also associated with synovitis. Exp Dermatol. 2004;13:347–356
5. Brown CL, Meise KC, Plowman GD, Coffey RJ, Dempsey PJ. Cell surface ectodomaine cleavage of human amphiregulin
precursor is sensitive to a metalloproteinase inhibitor. Release of a predominant N-glycosylated 43-KDa soluble form. J Biol Chem. 1998;273:17258–17268
6. Chung E, Cook PW, Parkos CA, Park YK, Pittelkow MR, Coffey RJ. Amphiregulin
causes functional downregulation of adherens junctions in psoriasis
. J Invest Dermatol. 2005;124:1134–1140
7. Pummi K, Malminen M, Aho H, Karvonen SL, Peltonen J, Peltonen S. Epidermal tight junctions: ZO-1 and occluding are expressed in mature developing and affected skin and in vitro differentiating keratinocytes. J Invest Dermatol. 2001;117:1050–1058
8. Zhou S, Matsuyoshi N, Takeuchi T, Ohtsuki Y, Miyachi Y. Reciprocal altered expression of T- cadherin and P- cadherin in psoriasis
vulgaris. Br J Dermatol. 2003;149:268–273
9. Liou A, Elias PM, Grunfeld C, Feingold KR, Wood LC. Amphiregulin
and nerve growth factor expression are regulated by barrier status in murine epidermis. J Invest Dermatol. 1997;108:73–77
10. Stoll S, Garner W, Elder J. Heparin-binding ligands mediate autocrine epidermal growth factor receptor activation in skin organ culture. J Clin Invest. 1997;100:1271–1281
11. Cook PW, Piepkorn M, Clegg CH, Plowman GD, DeMay JM, Brown JR, et al. Transgenic expression of the human amphiregulin
gene induces a psoriasis
-like phenotype. J Clin Invest. 1997;100:2286–2294
12. Johnston A, Arnadottir S, Gudjonsson JE, Aphale A, Sigmarsdottir AA, Gunnarsson SI, et al. Obesity in psoriasis
: leptin and resistin as mediators of cutaneous inflammation. Br J Dermatol. 2008;159:342–350
13. McCarty MF. Glucosamine for psoriasis
?. Med Hypotheses. 1997;48:437–441
14. Jemec GB, Wulf HC. The applicability of clinical scoring system: SCORAD and PASI in psoriasis
and atopic dermatitis. Acta Derm Venereol. 1997;77:392–393
15. Billings SD, Southall MD, Li T, Cook PW, Baldridge L, Moores WB, et al. Amphiregulin
overexpression results in rapidly growing keratinocytes tumors: an in vivo xenograft model of keratoacanthoma. Am J Pathol. 2003;163:2451–2458
16. Berasain C, Castillo J, Purgorria MJ, Prieto J, Avila MA. Amphiregulin
: a new growth factor in hepatocarcinogenesis. Cancer Lett. 2007;254:30–41
17. Yoshida A, Kanno H, Watabe D, Akasaka T, Sawai T. The role of heparin-binding EGF-like growth factor and amphiregulin
in the epidermal proliferation of psoriasis
in cooperation with TNF α. Arch Dermatol Res. 2008;300:37–45
18. Takahashi H, Tsuji H, Hashimoto Y, Ishida Yamamoto A, Lizuka H. Serum cytokines and growth factors levels in Japanese patients with psoriasis
. Clin Exp Dermatol. 2010;35:645–649