Brice Jason Williams and Dr. Allan M. Judd, Zoology
In most mammals, the adrenal cortex produces the hormones cortisol, aldosterone, and dehydroepiandrosterone (DHEA). These hormones have profound effects upon water and salt balance, the inflammatory response, and the metabolism of sugars, fats, and proteins. Because of its importance in maintaining homeostasis, the body highly regulates the adrenal gland. As part of the regulation, the body secretes many different molecules including cytokines. One of the most researched cytokines is interleukin-6, IL-6 (1). Many different cells produce IL-6, including adrenal cortical cells (2). The adrenal cortical cells secrete IL-6 into the extracellular space. IL-6 then binds to its receptor (IL-6 receptor or IL-6R). The specific pathway by which this cytokine activates gene production has been elucidated in other cell types, but the mechanism of IL-6- induced cortisol release is unknown (3).
Assuming that the IL-6 signaling pathway in the adrenal gland must be similar to the pathway in other cell types, i.e. Jak/Stat, we studied the phosphorylation of Janus Kinase 1 (Jak1), Jak2, and Tyrosine kinase 2 (Tyk2) and Stat 1 and Stat3 (Signal transducer and activator of transcription) phosphorylation in response to IL-6. The purpose of this study was 1) to show that the IL-6 pathway in the adrenal gland utilizes the method outlined and 2) to identify the specific Jak/Stat proteins used.
To accomplish this Bovine adrenal glands were obtained from an abattoir, the glands were sliced (1 mm thick) utilizing a tissue slicer, and zona reticularis and zona fasciculata separated from the surrounding adrenal tissue. The rings of adrenal tissue was then diced into 3 mm sections and incubated (5 minutes to 60 minutes) with RPMI culture medium containing vehicle or IL-6 (1 pg/mL – 100 ng/mL). The tissue fragments were than homogenized and the whole cell lysate exposed to precipitating antibodies against Jak1, Jak2, tyrosine kinase 2 (Tyk2), Stat1, or Stat3. Whole cell lysates were also obtained by culturing H295R cells (a human adrenal cell line).
The precipitated proteins were separated by electrophoresis and transferred to nitrocellulose membranes. The membranes were then exposed to phosphotyrosine antibodies and the binding of the antibodies to the proteins detected by a chemiluminescence assay. IL-6 increased the tyrosine phosphorylation of Jak2 but affected the phosphorylation of neither Jak1 nor Tyk2. Jak2 was phosphorylated in a time dependent manner with effects seen at 15, 30, and 60 minutes. Maximal Jak2 phosphorylation was observed at 30 minutes. The phosphorylation of Jak2 was dependent upon the IL-6 concentration with phosphorylation apparent at 1 pg/mL and increasing through 100 ng/mL IL-6.
IL-6 also increased the tyrosine phosphorylation of Stat1 and Stat3 with Stat3 phosphorylation much stronger than Stat1 phosphorylation. Stat3 phosphorylation was also observed in a time dependent manner with phosphorylation strongest at 30 minutes IL-6 exposure.
In summary, IL-6 increases the phosphorylation of Jak2 in a time and concentration dependent manner and also increases the phosphorylation of Stat1 and Stat3 in a time dependent manner. Therefore, IL-6 is probably increasing cortisol release from adrenal cells through the Jak2/Stat1- Stat3 pathway.
References
- M. Ehrhart-Dornstein and G. Vinson 1998. Intraadrenal Interaction in the Regulation of Adrenocortical Steroidogenesis, Endocrine Reviews 19, 101-143.
- A. M. Judd 1998. Cytokine Expression in the Rat Adrenal Cortex, Horm. Metab. Research 30, 404-410.
- P. Heinrich and L Graeve 1998. Review Article, Interleukin-6-type cytokine signaling through the gp130/JAK/Stat pathway, Biochem Journal 334, 297-314.