D Craig Seager and Dr. Allan M Judd, Physiology and Developmental Biology
The secretions of the adrenal gland are responsible for the body’s response to stress. One of the major mechanisms by which these secretions are regulated is by the way of the hypothalamuspituitary- adrenal axis (HPA axis). However, it was suspected that other mechanisms exist that are utilized by the body in order to up or down regulate adrenal secretions. One adrenal secretion, cortisol, is of special interest due to its great abundance and various effects in the body. Cortisol levels are generally regulated by ACTH, which is released by the pituitary. However, it was also shown to be increased by interleukin-6 by Allan Judd in 2000. Even though it has been shown that interleukin-6 greatly increases the level of cortisol from the adrenal gland, the method by which it occurs has been unclear. This research project hypothesized that interleukin-6 increases cortisol release via the cytokines c-fos and c-jun. C-fos and c-jun together form a common transcription factor name AP-1.
First, the effects of IL-6 on c-fos were studied. It was shown that IL-6 and ACTH increase c-fos transcription in adrenal samples. Figure one shows an increase in the levels of transcription in bovine adrenal tissue samples of the c-fos gene mediated by both IL-6 and ACTH. The gel shown was run on an agarose gel. The lanes represented are as follows: (Lanes A-D were taken from PCR samples formed by a c-fos primer. The thicker bands on top are primer-dimers bands, while the bands on the bottom are the c-fos bands. ) A: Untreated (control), B: IL-6, C: ACTH, D: IL-6/ ACTH. (Lanes E-H are 18S controls) E: Untreated, F: IL-6, G: ACTH, H: IL-6/ACTH. The DNA recovered from the PCR trials were sequenced by the BYU DNA Sequencing center. The samples submitted to sequencing matched the known c-fos sequence by 98%. In addition, the bands shown on the agarose gel above demonstrated the correct molecular weight of 223 base pairs.
Second, the effects of IL-6 on c-jun were studied. It was shown that IL-6 increases c-jun transcription in Adrenal Cells. Figure two demonstrates an increase in c-jun transcription in adrenal tumor cells (H295R cells) that resulted from their exposure to IL-6. The lanes represented are as follows: (Lanes A-D were taken from PCR samples formed by a c-jun primer. The thicker bands on top are primer-dimers bands, while the bands on the bottom are the c-jun bands.) A: IL-6/ cAMP, B: cAMP, C: IL-6, D:Untreated (control); (Lanes E-H are 18S controls) E: IL-6/ cAMP, F: cAMP, G: IL-6, H: Untreated.
As demonstrated above, at this point it appears that IL-6 does increase the transcription rates of c-fos and c-jun. However, the next step in my research will be to further verify the data collected by replicating the experiments that have already been performed. The experiments above have had preliminary replication, but further replication is needed to add strength to the results that I reported. C-fos will also need to be measured in H295R cells, while c-jun needs to be measured in bovine adrenal tissue samples. Again this will be done to increase the validity of the research.
After replication is accomplished, the cells will be introduced to dominant negative mutants of both c-fos and c-jun in order to determine the relationship between AP-1 and cortisol release. Northern blots, run-on assays, and a time course, in order to establish the relationship between the increase in c-fos and c-jun transcription and cortisol release, will also be ran.
This research is very applicable and important to medicine. Similar studies have indicated that the HPA axis plays an important role in the susceptibility and resistance to inflammatory and infectious diseases by demonstrating the association between a blunted HPA response and an enhanced susceptibility to inflammation (Jafarian-Tehrani 2000). These studies indicate that cortisol, a secretion of the adrenal gland, is actively involved in the body’s response to stress and inflammation. Furthermore, inadequate levels of cortisol in the body have been shown to lead to severe autoimmune and inflammatory diseases. Examples of such complications due to low levels of cortisol include rheumatoid arthritis, asthma, thyroiditis, hay fever, encephalomyelitis, and many others.