Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that have a pleiotropic impact on the regulation of differentiation, cell growth, and metabolism of lipids and glucose 1. They exist in three different isoforms termed PPAR α, PPAR β/δ, and PPAR γ. PPARs are expressed during normal skin development in the epidermis, hair follicles, and sebaceous glands 2. Ligand activation of PPAR β/δ induces terminal differentiation of epithelium and other cells 3, and this is typically associated with concomitantly increased apoptotic signaling and/or decreased cell proliferation of keratinocytes, fibroblasts, and endothelial cells 4. Controversially, increased PPAR β/δ expression has been observed in premalignant actinic keratoses and overt squamous cell carcinoma 5,6. In another study, a novel PPAR δ antagonist, SR13904, was found to have antiproliferative activity in human cancer cells 7. Accordingly, the role of PPAR β/δ in cell growth remains controversial; some studies have found that upregulating PPAR β/δ expression leads to antiapoptotic signaling and increases cell proliferation, whereas others have found that activating PPAR β/δ inhibits cell proliferation by inducing terminal differentiation and/or proapoptotic signaling 4. Cutaneous T-cell lymphomas (CTCL) are a group of lymphoproliferative disorders characterized by localization of malignant T lymphocytes to the skin. Mycosis fungoides (MF), the most common and indolent form of CTCL, is characterized by patches, plaques, or tumors containing epidermotropic CD4+CD45RO+ helper/memory T cells 8, in which dysregulated apoptosis of skin-homing memory T cells is involved in the pathogenesis and therapeutic implications 9.
This work was conducted to detect PPAR β/δ expression in early cases of MF and compare it with that in normal healthy controls, in an attempt to define its role in MF pathogenesis.
Patients and methods
The study was approved by the Dermatology Research Ethical Committee (DermaREC) of the Faculty of Medicine, Cairo University, a representative of the institutional Research Ethical Committee. Informed written consent was signed by each individual before inclusion in the study. The study included 44 individuals, 20 patients diagnosed with early MF (stages Ia, Ib, and IIa) and 24 healthy controls.
Each patient was subjected to complete history taking and a full clinical examination to detect the extent and type of MF; the diagnosis was confirmed by histopathological examination. Staging of MF was further completed by lymph node examination and biopsy (when indicated) in the surgical outpatient clinic, Faculty of Medicine, Cairo University. Laboratory investigations (analyses of complete blood profile, liver and kidney function tests, fasting and 2-h postprandial blood sugar levels, and lactate dehydrogenase levels) and radiological investigations (chest radiograph and abdominal ultrasound) were carried out.
Twenty-four age-matched and sex-matched healthy volunteers with no history of skin or autoimmune diseases served as controls. A 4-mm punch skin biopsy was obtained from each patient, stored at −70°C, and analyzed semiquantitatively for the level of PPAR β/δ mRNA using the reverse transcriptase-PCR technique.
Quantitation of the PCR product
RNA was extracted from tissues using an RNA extraction kit. Reverse transcriptase-PCR analysis of PPAR β/δ was carried out on extracted RNA using specific primers with sequences of sense 5-TCCCTCTTTCTCAGTTCCTC-3 and antisense 5-CAGGAGACAGAAGTGAGGAC-3 10. A total of 5 µg was extracted from skin biopsies and denaturized for 10 min at 70°C; then, RNA was reverse transcribed into cDNA using SuperScript II RNase H-reverse transcriptase (5 U per reaction; Invitrogen, Groningen, the Netherlands). The cDNA was subjected to PCR amplification under the following conditions: 5 min denaturation at 94°C, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, with final extension for 7 min at 72°C. The PCR mixture contained 1× buffer, oligonucleotide primers, dNTPs, 1 U of Taq DNA polymerase, and cDNA. In every case, a PCR negative control (without DNA) was included. PCR products were electrophoresed on 2% agarose gels stained with ethidium bromide. Positive bands appeared at 287 bp. PCR products were then quantitated using a quantitation kit (Promega Corporation, Madison, Wisconsin, USA). This method depends on purification of the PCR using a Promega Wizard PCR preps DNA purification kit (Promega Corporation). The mixture for quantitation consisted of DNA quantitation buffer, sodium pyrophosphate, NDPK enzyme solution, T4 DNA polymerase, and DNA. All these contents were incubated at 37°C for 10 min. Thereafter, 100 μl of Enliten L/L reagent was added. The reaction was read immediately using a luminometer. The same steps were carried out on DNAs of known concentrations provided by the kit, and a standard curve was plotted using the readings of the luminometer against the concentrations. Thereafter, the readings of the amplified PCR product of PPAR β/δ after using the luminometer were read from the standard curve. The results were expressed as pg/g tissue 11.
Data were analyzed using Statistical Program of Social Sciences (SPSS) for windows version 11 (SPSS Inc., Chicago, Illinois, USA). Data were represented as mean±SD for numerical data or as numbers and percentages for non-numerical data. They were compared using an independent t-test (for parametric quantitative data) or the Mann–Whitney U-test (for nonparametric quantitative data). The different variables were correlated using Pearson’s correlation coefficient (for numerical data). P value less than 0.05 (two-tailed) was considered significant.
This study included 20 MF patients [15 male (75%) and five female (25%)] and 24 controls [13 male (54.2%) and 11 female (45.8%)]. The patients with MF ranged in age from 9 to 30 years, with a mean of 18.75±6.20 years, and their disease duration ranged from 4 to 108 months, with a mean of 32.15±26.6 months. The extent of lesions ranged from 5 to 90% of body surface area (mean of 51±26.03%). The healthy volunteers ranged in age from 16 to 50 years, with a mean of 27.04±9.08 years (Table 1).
Levels of PPAR β/δ mRNA ranged from 601 to 2782 µg/g, with a mean of 1331.7±659.72 µg/g, in patients with MF and from 314 to 874 µg/g, with a mean of 633.33±131.65 µg/g, in controls. MF patients showed significantly higher levels of PPAR β/δ than did controls (P <0.001) (Table 2, Fig. 1).
PPAR β/δ levels showed a significant positive correlation with the extent of lesions in MF patients (P=0.008) (Fig. 2), whereas the correlation was not significant with either age or disease duration (P=0.23 and 0.80, respectively) (Table 3).
There is considerable controversy on whether PPAR β/δ stimulates or inhibits cell proliferation. Activation of PPAR β/δ disturbs normal cell physiology depending on the cell type, and, regardless of the consequences of PPAR β/δ activation, it seems to control the inflammatory switch and balance between cell survival and growth. Therefore, disruption of PPAR β/δ might lead to uncontrolled cell growth 12, and its activation has been shown to increase human cancer growth in many tissues including liver, colon, breast, prostate, and lung 13,14; however, contrasting results have also been observed 15–17. It can be speculated that PPAR β/δ agonists display a variety of effects on protumor and antitumor processes and seem to have proangiogenic activity at very low concentrations 1.
Results of the present study showed a significant increase in PPAR β/δ expression in skin lesions of MF patients and also showed a significant positive correlation of PPAR β/δ levels with the extent of lesions in these patients. To our knowledge, this the first study investigating PPAR β/δ expression in MF patients. This high expression of PPAR β/δ in our patient group of early MF may be rationalized by the display of an antiapoptotic effect by PPAR β/δ on malignant T cells, up to a probable role in the potentiation of T-cell proliferation. Various molecular events have been implicated in the promotion of carcinogenesis by PPAR β/δ, including induction of COX-2 expression and prostaglandin E2 (PGE2) production 14 and PGE2 receptor subtype EP4 expression through phosphatidylinositol-3 kinase signaling 18. COX-2-dependent PGE2 acts as a growth factor in MF 19. Further, PPAR β/δ causes increased expression of vascular endothelial growth factor 20. Malignant CTCL T cells were found to be spontaneously producing the potent angiogenic protein, vascular endothelial growth factor 21. PPAR β/δ causes reduction of the proapoptotic cell death-inducing DFF45-like effector A and B levels, increase of the antiapoptotic inhibitor of caspase-activated deoxyribonuclease short and long forms (ICAD-S and ICAD-L) 22, activation of the antiapoptotic Akt1 pathway 23, and increase in nuclear factor κB activation and matrix metalloproteinase-9 secretion 24. In early stages of CTCL, the transcription factor nuclear factor κB has been shown to be constitutively active in malignant T cells, in which it promotes proliferation and cell survival 25. Also, the general intensity of expression of MMP-9 was found to be significantly higher in patients with MF than in controls, and it increased in direct proportion to the increase in disease severity, being greatest in the tumor stages 26. However, our study has focused on the expression of the PPAR β/δ receptor rather than on ligand activation; it did not demonstrate increased transcriptional activity of target genes due to the relative presence of PPAR β/δ. In the absence of these data, it is likely that the described increase in PPAR β/δ mRNA is not necessarily reflected at the protein and/or functional level. Therefore, direct evidence of alterations in well-characterized PPAR β/δ target genes should be sought while examining the effect of PPAR β/δ on cell growth as proposed earlier 4.
PPAR β/δ mRNA levels showed a positive correlation with the extent of MF lesions, which suggests that PPAR β/δ levels could be considered a reflection of disease severity. In contrast, PPAR β/δ did not show any significant correlation with disease duration in MF patients, suggesting its role in initiation but not in maintaining the disease process.
In conclusion, our study documents the possible role of PPAR β/δ in the pathogenesis of MF and raises its contribution to the severity of the disease. Clarification of the functional role of PPAR β/δ through more standardized studies is needed.
The authors express their gratitude to Dr Wedad Mostafa from the Dermatology Department, Cairo University, whose help, suggestions, and encouragement helped them at the time of research for writing this paper.
Conflicts of interest
There are no conflicts of interest.
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