Keratinocyte CGRP Expression and Its Potential Contribution to Chronic Pain

Calcitonin gene related peptide (CGRP) plays an important role in pain, and is also involved in the pathogenesis of skin diseases such as psoriasis. All the known species of CGRP have 37 amino acids and occur in two isoforms, alpha and beta. The two isoforms are translated from two different genes, but have a nearly identical structure and bind to the same receptor complex with similar affinity. They are typically co-expressed in the same types of cells and are assumed to mediate the same functions, although there are a few functionally specific locations where only one isoform is expressed. The source of most CGRP in our body is presumed to be released from the axon terminals of neurons that predominantly produce and release CGRPalpha. However, our research on the skin has repeatedly detected CGRP-like immunofluorescence in epidermal keratinocytes as well as sensory innervation in a variety of mammalian species ranging from mice to humans. I hypothesized that epidermal keratinocytes produce CGRP, that CGRP levels in keratinocytes can be regulated under various normal and pathological conditions, and that CGRP can be released from keratinocytes upon noxious stimulation and chronic pain conditions. Both skin tissues and cultured keratinocytes were used in the study. RT-PCR, qPCR, and gene array confirmed for the first time the expression of CGRP mRNA in keratinocytes, while immunohistochemical staining and western blot confirmed the presence of CGRP peptide. Enzyme immunoassay (EIA) was used to document the release of CGRP from keratinocytes under noxious heat stimulation. Surprisingly my research revealed that the epidermal keratinocytes make predominantly CGRPbeta, whereas prior studies documented that neurons predominantly produce CGRPalpha. The mRNA level of CGRPbeta was about 3.5 and 10 folds higher than that of CGRPalpha in mice and human keratinocytes, respectively. Compared to their controls, elevated levels of CGRP immunoreactivity were detected in epidermal keratinocytes in the painful skin of humans afflicted with complex regional pain syndrome (CRPS) and postherpetic neurogia (PHN), in monkeys infected with SIV, and in rats with prolonged pain induced by subcutaneous injection of CFA and by constriction and chronic nerve injury. CGRP immunoreactivity was also increased in the epidermal keratinocytes in mice having K14 promotor driven transgenic over-expression of the BMP4 antagonist noggin. Laser capture and qPCR analyses of the epidermis in the noggin-overexpressing mice confirmed a significantly higher level of CGRP mRNA than in mice overexpressing BMP4. In addition, CGRP release was observed from mouse keratinocytes following noxious heat (48°C) stimulation. These results support the hypothesis that CGRP is produced by epidermal keratinocytes and is released under noxious thermal stimulation. The keratinocytes produce predominantly the beta isoform of CGRP, in contrast to neurons that predominantly produce CGRPalpha. Elevated levels of CGRP immunoreactivity in epidermal keratinocytes in humans with CRPS and PHN and in animal models of induced prolonged pain suggest that keratinocyte derived CGRPbeta may be a major contributor to chronic pain.