InsulinLike Growth Factor Receptor 1 Gene Expression in Psoriasis
Abstract
Background: Keratinocyte proliferation and differentiation are controlled by a complex network of several growth factors such as insulin-like growth factors.
Objectives: The aim of this study was to assess quantitatively the expression of insulinlike growth factor receptor 1 (IGFR1) gene in psoriasis.
Method: Nineteen cases with plaque stage psoriasis vulgaris and ten controls were enrolled into this study. A 3 mm punch biopsy specimen was obtained from each patient and control. Detection of IGFR-1 gene expression was done by Reversetranscriptionpolymerase chain reaction (RTPCR).
Results: The expression of IGFR1gene was elevated in psoriatic patients and this level was statistically higher compared to controls.
Conclusion: The increased expression of IGFR1 gene in lesional psoriatic skin could be important in regulating the keratinocyte hyperplasia associated with this disorder. Further studies are needed to highlight the role of IGFR1 in psoriasis.
Introduction
Psoriasis is a common inflammatory skin disease with lesions characterized by keratinocyte hyperproliferation and infiltration of activated leukocytes in the skin. The etiology of keratinocyte hyperproliferation found in psoriasis is not well understood. Immunological pathomechanisms had been favored. For example, intercellular mediators released from activated T-cells, cytokines and growth factors released from keratinocytes and/or dermal fibroblasts(1-9). Keratinocyte proliferation and differentiation are controlled by a complex network of several growth factors including insulinlike growth factors (IGFs) and transforming growth factorα (TGFα)(10,11).
Insulin-like growth factors (IGFs) (somatomedins), which were initially identified as potent physiological mitogens, are family of growth hormonedependent polypeptides that exert effects on vatiety of cells’ proliferation, differentiation, apoptosis and transformation(12). Two human somatomedins, Insulin- like growth factor І (IGFI) and Insulin-like growth factorII (IGFII) have been identified(13). IGFs can induce their effect through interactions with unique cell surface receptors. IGF-І is a 70 amino acid, 7.5 kD peptide that has approximately 50% structural homology to proinsulin, and is detectable in most tissues. IGFI is ascribed the function of growth by acting through the Insulin-Like Growth Factor Receptor-I (IGFR1) which is found on most cells(13,14). IGFR1 is composed of two α subunits; each is a 125kD protein that is entirely extracellular and functions as ligand binding, and two ß subunits; each is a 95 kD transmembrane protein, with extracellular and cytoplasmic domains. These chains are linked in a heterotetrameric disulfidelinked complex(13). IGFI is an important factor for the growth and function of both keratinocytes and fibroblasts in the skin(15). IGF-ІІ can influence mitogenesis in a variety of cell types, including normal and transformed human keratinocytes(16).
Previous studies found that the level of IGFs was elevated in the serum in psoriatic skin. However, the specific molecular function of these factors and their receptors in promoting psoriatic hyperplasia remains to be investigated(12,17,18). Also, these findings did not tell whether this high level of expression of IGFs and their receptors is due to genetic factor or as a result of other factors. So, the aim of this study was to evaluate the expression of IGFR1 gene quantitatively in psoriatic skin and to compare it to normal controls in a trial to open the way for more researches on IGFs and their receptors in psoriasis.
Patients and Method
The study was conducted in 2010 in the Outpatient Dermatology Clinic in Kasr El Aini hospital. The study was conducted on 19 psoriatic patients. The inclusion criteria were stable, bilateral and symmetrical plaque stage psoriasis vulgaris without topical or systemic treatment 6 weeks prior to the study. Clinical assessment was done for each patient and involved body surface area was evaluated by Rule of Nines. Ten healthy volunteers had been enrolled as controls; 6 females and four males. Their ages ranged from 20 to 52 years old. A 3-mm punch skin biopsy specimen was taken from the lesional skin of each psoriatic patient and control. Detection of IGFR1gene expression was done by Reverse transcriptionpolymerase chain reaction (RTPCR) technique.
RNA extraction:
Total RNA was extracted from skin tissue by the acid guanidinum thiocyanate phenol chloroform method(19). RNA content and purity were measured by a UV spectrophotometer.
RTPCR experiments:
RTPCR was done using the extracted RNA for detection of IGF receptor gene. For amplification of the targets gene, reverse transcription and PCR were run in two separate steps. Briefly, Reaction mixture of RT reaction containing 1 μg total RNA, 0.5 μg random primer, 5×RT buffer, 2.5 mmol/L dNTP, 20 U RNase inhibitor and 200 U MMLV reverse transcriptase in a total volume of 25 µl was incubated at 37ºC for 60 minutes, then heated to 95ºC for 5 minutes to inactivate MMLV.
PCR was carried out with 1.5 μl RT products, 10 × PCR buffer (without Mg2+) 2.5 μl, 2.0 μl dNTP (2.5 mmol/L), 2.0 μl MgCl2 (25 mmol/L), 0.5 μl each primer (20 μmol/L) of β-actin, 0.5 μl each primer of gene to be tested (20 μmol/L) and 1 U of Taq DNA polymerase (Promega Corp.), in a final volume of 25 μl. Thermal cycler conditions were as follows: a first denaturing cycle at 97ºC for 5 min, followed by a variable number of cycles of amplification defined by denaturation at 96ºC for 1.5 min, annealing for 1.5 min, and extension at 72ºC for 3 min. A final extension cycle of 72ºC for 15 min was included. The appropriate primer sequence of IGF receptor Forward primer: CTGGAAACCTGATGGGTGGTC, Reverse primer: CAACAGCTGAAAGAACGCCCTAgarose gel delectrophoresis:All PCR products were electrophoresed on 2% agarose stained with ethidium bromide and visualized by UV transilluminator.
Semi-quantitative determination of PCR products:
Semiquantitation was performed using the gel documentation system (BioDO, Analyser) supplied by Biometra. According to the following amplification procedure, relative expression of each studied gene (R) was calculated following the formula:
R= Densitometrical Units of the studied gene/ Densitometrical Units of b-actin.
Statistics
Descriptive statistics were performed for all variables of the study. For quantitative variables, the mean range, standard deviation (± SD) and standard error of the mean (± SEM) were calculated. For categorical variables absolute counts as well as percentages were generated. Comparison of quantitative data was tested using “Student t-test” to compare two groups, or a non-parametric (Mann Whitney/ Wilcoxon) test whenever appropriate comparison of categorical data was done using the Chi-square test. Correlation study for relationship of different variables was done using Spearman rank order correlation “R”.P value will be considered significant if <0.05. The statistical program used was SPSS, Illinois, Chicago, USA.
Results
This study was conducted on 19 cases of psoriasis vulgaris and ten normal controls. Patients’ data is illustrated in Table (1). Ten controls; 60% were females and 40% were males. Their mean age was 40.30 ± 10.46 years old. The levels of IGFR1gene expression in psoriatic patients and controls were illustrated in Table (2) and Figs (1 & 2). Comparison between psoriatic patient and controls regarding IGFR1 gene expression showed high statistically significant difference, being higher in patients than controls (P value < 0.001). The level of IGFR1 gene expressions in psoriatic patients’ subgroups (as regards sex of the patients and localization of the lesions) were done and illustrated in Table (3). No significant difference was found between males and females (P value>0.05) (Fig. 3), generalized and localized lesions (P>0.05) (Fig. 4) as regards IGFR1 gene expression. There was no statistically significant correlation between the level of IGFR1 gene expression and each of age of the patients (P>0.05), disease duration (P>0.05) and percentage of the involved body surface area (P>0.05).
Table 1. Clinical data of psoriatic patients.
Variables |
Data |
Sex of the patients: Females Males |
11 patients (57.9%) 8 patients (42.1%) |
Age of the patients (years): Range Mean ± SD |
1468 41.00 ± 17.61 |
Duration of the disease (years): Range Mean ± SD |
0.7535 8.41 ± 1.80 |
Distribution of the lesions: Localized Generalized |
2 patients (10.53%) 17 patients (89.47%) |
Percentage of the involved total body surface area (Rule of Nine): Range Mean ± SD |
20%80% 54.21 ± 18.35 |
Past history of hypertension: |
1 patient (5.26%) |
Family history of: Psoriasis Diabetes Mellitus |
2 patients (10.53%) 4 patients (21%) |
SD: Standard deviation
Table 2. Levels of insulin-like growth factor receptor 1 (IGFR1) gene expression in psoriatic patients and controls.
|
Psoriatic patients |
Controls |
Range |
0.53-3.07 |
0.12-1.04 |
Mean ± SD |
1.32 ± 0.67 |
0.34 ± 0.27 |
SD: Standard deviation
Table 3. Relations between the levels of insulinlike growth factor receptor 1 (IGFR1) gene expression in different subgroups of psoriatic patients.
Variables |
Range |
Mean ± SD |
Sex of the patients: Females Males |
0.532.13 0.54-3.07 |
1.20 ± 0.58 1.48 ± 0.79 |
Psoriatic lesion: Localized Generalized |
0.53-1.30 0.54-3.07 |
0.92 ± 0.54 1.36 ± 0.68 |
SD: Standard deviation
Discussion
Psoriasis is a common chronic skin disease characterized epidermal hyperproliferation. The underlying cause of the aberrant keratinocyte growth control is thought to be the presence of activated T lymphocytes at the dermal/epidermal interface or due to expression of several growth factors and cytokines(10,18,19,20). IGF-I demonstrates a potent mitogenic action on the epidermal cells. It is produced by dermal fibroblasts, epidermal melanocytes, and keratinocytes within the stratum granulosum and acts through the IGFRI which is expressed in the basal layer of the epidermis(21,22), the outer root sheath, hair matrix cells, sebaceous gland, eccrine sweat duct, and myoepithelial cells of the secretory portion of eccrine sweat gland. IGFR1 expression occurred principally in undifferentiated epithelial cells in the epidermis and skin appendages(23). Expression of IGFR1 in sweat duct epithelium and myoepithelial cells, which are metabolically very active but mitotically inactive, indicates that IGFR1 serves not only as a mitogenic factor, but also as a factor regulating the synthetic activity of a variety of cells(24). Many studies were done to investigate the roles of IGFI and its receptor in regulation of skin structure and function. In connective tissue cells, such as fibroblasts, IGFI regulates cell proliferation in conjunction with plateletderived growth factor and other mitogens. In keratinocytes, IGFI regulates proliferation via synergistic interactions with epidermal growth factor (EGF) or fibroblast growth factor (FGF)(25,26,27). Expression of IGF1 and its receptors beyond the basal layers was found in the hyperproliferative skin disorders such as psoriasis, acanthotic epidermis of the edge of stasis ulcers, and the acanthotic epidermis of plaque stage mycosis fungoides which correlated with expression of the hyperproliferative keratin K16(23). Also, IGFR1 appears to mediate hyperresponsiveness of epidermal cells to other cytokines(28).
In the present study, a high level of expression of IGFR1 gene in psoriatic skin was found. There was a significant difference between IGFR1 gene expression in psoriatic skin compared to normal controls. Unfortunately, this high level of secretion in psoriatic skin had no significant relation with age of the patients, sex of the patients, duration of the disease and extent of total body surface area involved by psoriasis. Actually, we could not find studies to agree with or contradict our results; because it is an early attempt to evaluate the level of IGFR1 gene expression in psoriatic skin. Previous researches studied the presence and location of IGFR1 itself in psoriatic skin using staining method while in our study we estimate the amount of IGFR1 gene expression quantitatively in psoriasis using RT-PCR method. The high level of secretion of IGFR1 gene in psoriasis aroused many questions as regards its role in the pathogenesis of psoriasis and if this has relation to metabolic syndromes associated with psoriasis especially Diabetes mellitus . The pathway by which this receptor is transmitting its unique signal and the diverging point on the shared receptors between insulin and IGF1 needs to be searched for in future. Another question; could the level of IGFR1gene expression be one of predicting factor for patient’s course of the disease, its severity, prognosis and response to treatment. The answers to all these questions need further studies and more prolonged follow up of psoriatic patients.
Overall, our results clearly demonstrate that IGFR1 gene is distinctly unregulated during keratinocyte hyperproliferaton in psoriasis. This might help in future treatment strategies for this disease.
References
1. Fry L (1988). Psoriasis. Br J Dermatol;199:445461
3. Paukkonen K, Naukkarenen A, Horsmanheimo M (1992). The development of manifest psoriatic lesions is linked with the invasion of CD8+ T cells and CD11c+ macrophages into the epidermis. Arch Dermatol Res; 284:375389
3. Bos J (1988). The pathomechanisms of psoriasis: the skin immune system and cyclosporine. Br J Dermatol; 118: 141155
4. Lui S and Parsons C (1983). Serial cultivation of epidermal keratinocytes from psoriasis plaques. J Invest Dermatol; 81:5461
5. Ristow H (1997). Increased synergistic effect of EGF and IGFI on DNA synthesis of cultured psoriatic keratinocytes. Dermatology; 195: 213219.
6. Fraki J, Briggaman R, Lazarus G (1983). Transplantation of psoriatic skin onto nude mice. J Invest Dermatol; 80: 3135.
7. Saiag P, Coulomb B, Lebreton C, Bell E, Dubertret L (1985). Psoriatic fibroblast induces hyperproliferation of normal keratinocytes in a skin equivalent model in vitro. Science; 230: 669672.
8. Krueger G and Jorgensen C (1990). Experimental models for psoriasis. J Invest Dermatol; 95: 56S58S.
9. Mura H, Sano S,Higashiyama M and Yoshikawa K (2000). Involvement of insulin like growth factor 1 in psoriasis as a paracrine growth factor. Arch Dermatol Res; 292: 590597.
10. Ikai K (1999). Psoriasis and the arachidonic acid cascade. Journal of Dermatological Science; 21: 135146.
11. Nickoloff B (1999). The immunologic and genetic basis of psoriasis. Archives of Dermatology; 135: 11041110.
12. Yoo H, Kim S, Kim Y, Lee H and Kim T (2007). Insulin like growth factor-II regulates the 12 lipoxygenase gene expression and promotes cell proliferation in human keratinocytes via the extracellular regulatory kinase and phosphatidyl inositol 3kinase pathway. The international J of Biochemistry and Cell Biology; 39: 12481259.
13. Misra P, Nickoloff B, Morhenn V, Hintz R and Rosefeld R (1986). Characterization of Insulin-like Growth Factor-I/ Somatomedin-C Receptors on human keratinocyte Monolayers. J Invest Dermatol; 87: 264267.
14. Swope V, Supp A, Greenhalgh D, Warden G and Boyce S (2001). Expression of Insulinlike growth factor I by cultured skin substitutes does not replace the physiologic requirement for insulin in vitro. J Invest Dermatol; 116: 650657.
15. Nicoloff B, Misra P, Morhenn V, Hintz R, Rosenfeld R (1988). Further characterization of the keratinocyte somatomedinC / insulinlike growth factor–I (SMC/IGF I) receptor and the biological responsiveness of cultured keratinocytes to SMC/IGF I. Dermatologica; 177: 265273.
16. Neely E, Morhenn V, Hintz R, Wilson D, Rosenfeld R (1991). Insulin-like growth factors are mitogenic for human keratinocyes and a squamous cell carcinoma. J Invest Dermatol; 96: 1041100.
17. Xu S, CwyfanHughes S, Van Der Stappen J, Sansom J, Burton J, Donnely M, et al. (1996). Altered insulinlike growth factorII (IGF II) level and IGF binding prtien3 (IGFBP3) protease activity in interstitial fluid taken from the skin lesion of psoriasis. J Invest Dermatol; 106: 109112.
18. Kwon Y,Kwon K, Moon H, Park J, Choi K, et al. (2004). Insulin-like growth factor-II regulates the expression of vascular endothelial growth factor by the human keratinocyte cell line Ha CaT. J Invest Dermatol; 123: 152158.
19. Chomkczynski P and Sacchi N (1987). Single step method for RNA isolation by the acid guanidinum thiocyanatephenolchloroform method. Analytical Biochemistry; 162: 156160.
20. Nickoloff B (1991). The cytokine network in psoriasis. Arch Dermatol; 127: 871884.
21. Gottlieb A (1997). Immunopathogenesis of psoriasis: the road from bench to bedside is a twoway street. Arch Dermatol; 133: 781782.
22. Sadowski T, Dietrich S, Koschinsky F and Sedlacek R (2003). Matrix Metalloproteinase 19 regulates insulin-like growth factor mediated proliferation, migration and adhesion in human keratinocytes through Proteolysis of insulinlike growth factor binding protein3. Molecular Biology of the Cell; 14: 45694580.
23. Krane J, Gottlieb A, Carter M and Krueger J (1992). The insulinlike growth factor I receptor is overexpressed in psoriatic epidermis, but is differentially regulated from the epidermal growth factor receptor. J Exp Med; 175: 10811990.
24. Hodak E, Gottlieb A, Anzilotti M, Krueger J (1996). The insulinlike growth factor 1 receptor is expressed by epithelial cells with proliferative potential in human epidermis and skin appendages: Correlation of increased expression with epidermal hyperplasia. J Invest Dermatol; 106: 564570.
25. Nanney L, Magid M, Stoscheck C, King L (1984). Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages. J Invest Dermatol; 83: 385393.
26. Krane J, Murphy D, Carter D and Krueger J (1991). Synergistic effects of epidermal growth factor (EGF) and insulin-like growth factor 1 / somato-medins C (IGFI) on keratinocyte proliferation may be mediated by IGFI transmodulation of the EGF receptor. J Invest Dermatol; 96: 419.
27. Nanney L, Stoscheck C, Magid M, and King L (1986). Altered epidermal growth factor binding and receptor distribution in psoriasis. J Invest Dermatol; 86: 260.
28. Ristow H (1997). Increased synergistic effect of EGF and IGFI on DNA synthesis of cultured psoriatic keratinocytes. Dermatology; 195: 213219.
29. Wraight C, Edmondson S, Fortune D, Varigos G and Werther G (1997). Expression of IGF binding protein3 (IGFBP3) in the psoriatic lesion. J Invest Dermatol; 108: 452456.