Effects of Protein Kinase C Down‐Regulation on Secretory Events and Proopiomelanocortin Gene Expression in Anterior Pituitary Tumor (AtT‐20) Cells

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Abstract
To elucidate the role of the diacylglycerol‐protein kinase C (PKC) pathway in β‐endorphin synthesis and secretion in anterior pituitary corticotrope tumor cells (AtT‐20), a procedure for down‐regulating PKC activity in the cells was developed. Treatment of AtT‐20 cells with 12‐0‐tetradecanoylphorbol 13‐acetate (TPA) led to an increase in [3H]phorbol 12,13‐dibutyrate binding to PKC in the membrane fraction of these cells 30 s after its addition to the culture medium. Thereafter, a decrease in both [3H]phorbol 12,13‐dibutyrate binding and PKC‐specific phosphotransferase activity occurred in a time‐ and dose‐dependent manner in both the cytosolic and membrane fractions. For example, treatment of the cells with 100 nM TPA for 24 h resulted in an almost complete depletion of PKC activity. Immuno‐reactive β‐endorphin secretion was found to be stimulated two‐ to fourfold in the control cells after incubation with corticotropin‐releasing factor (10‐7M), forskolin (10‐6 M), or TPA (10‐7M) for 4 h. In cells rendered PKC deficient, TPA‐stimulated immunoreactive β‐endorphin release was abolished, forskolin‐stimulated release was unaffected, and corticotropin‐releasing factor‐stimulated release was depressed. Treatment of control cells with any one of the three stimulatory agents led to an increase in proopiomelanocortin mRNA levels, and these responses were also depressed after TPA pretreatment. The results suggest that physiological processes thought to be entirely cyclic AMP dependent, such as corticotropin‐releasing factor‐elicited secretion, may be partially dependent on PKC‐mediated biochemical events.