Altered gene expression in melanocytes exposed to 4-tertiary butyl phenol (4-TBP): upregulation of the A2b adenosine receptor 1.
Exposure to phenolic agents contributes to the development of occupational vitiligo. Proposed as a causative factor for leukoderma in vivo, the para-substituted phenol 4-tertiary butyl phenol was chosen to investigate early cellular events responsible for selective disappearance of melanocytes from the epidermis of individuals sensitive to such agents. To this end, differential display of melanocyte mRNA isolated from three separate cultures was performed following a 12 h exposure of cells to 250 microM 4-tertiary butyl phenol or to vehicle alone. Fragments of cDNA representing differentially expressed messages were cloned and subsequently confirmed by reverse dot blotting. Alignment analysis revealed that the L30 ribosomal protein was upregulated by the treatment, potentially reflecting altered levels of protein synthesis in response to stress. In addition, a gene sequence upregulated following exposure to 4-tertiary butyl phenol was identified as the A2b receptor (a P1 receptor for adenosine). Differential expression of this gene was confirmed in an RNase protection assay. By reverse transcription-polymerase chain reaction, the gene was shown to be expressed in keratinocytes and fibroblasts as well. Flow cytometry confirmed differential expression in melanocytes and fibroblasts, but not in keratinocytes. Interestingly, it has been reported that P1 purinoceptor stimulation can induce apoptosis. This is in concordance with results reported elsewhere demonstrating induction of apoptosis by 4-tertiary butyl phenol in human melanocytes, as well as with morphologic changes observed in this study in cells exposed to 250 microM 4-tertiary butyl phenol for 72 h. In conclusion, differential display is useful to establish melanocyte components involved in the cellular response to phenolic agents.
Le Poole
I C
IC
Department of Pathology, Loyola University Strich School of Medicine, Maywood, Illnois 60153-5385, USA. ilepool@luc.edu
Yang
F
F
Brown
T L
TL
Cornelius
J
J
Babcock
G F
GF
Das
P K
PK
Boissy
R E
RE
D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D008544:Melanocytes / Q000378:metabolism*; D010636:Phenols / Q000494:pharmacology*; D010859:Pigmentation Disorders / Q000517:prevention & control; D043704:Receptor, Adenosine A2B; D018047:Receptors, Purinergic P1 / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000502:physiology; D017423:Sequence Analysis, RNA; D015854:Up-Regulation
D010636:Phenols; D043704:Receptor, Adenosine A2B; D018047:Receptors, Purinergic P1; D012260:Ribonucleases