Cytokine induction of heat shock protein in human granulosa-luteal cells

The infiltration of leukocytes is a characteristic feature of luteolysis in humans. Leukocytes are known to generate physiological inducers of cell stress such as cytokines which have been implicated as mediators of functional luteal regression. In cells exposed to stress, a response characterized by an increase in heat shock protein (HSP) synthesis occurs. Recently, the induction of HSP-70 in rat luteal cells has been shown to inhibit luteinizing hormone (LH) and cAMP-sensitive progesterone production, possibly by interfering with the translocation of cholesterol to the mitochondrial cytochrome P450scc. We therefore investigated whether HSP-70 is induced in human granulosa-luteal cells and its relationship to steroidogenesis. [35S]Methionine labelling showed an increase in a 70 kDa protein after heat treatment which was demonstrated to be HSP-70 by Western analysis using monoclonal antibodies against the constitutive and inducible forms of HSP-70. Induction of HSP-70 in human granulosa-luteal cells was also seen with interferon (IFN) gamma (10 ng/ml), tumour necrosis factor (TNF)-alpha (100 ng/ml) and a combination of IFN gamma/TNF-alpha (10/50 ng/ml). Interleukin-1 beta (IL-1 beta) (30 ng/ml) showed minimal induction of HSP-70 above control values. An increase in activated heat shock factor, which binds to the heat shock transcriptional control element, was detected after heat shock, IFN/TNF, and IFN treatment. Coincident with the induction of HSP-70 by heat shock was the inhibition of progesterone production compared with non-shocked granulosa-luteal cells. Heat shock inhibition of progesterone synthesis was partially reversed by the cell- and mitochondria-permeant cholesterol analogue, 22R-hydroxycholesterol. Cell viability was unaffected by heat treatment. White blood cell-depleted granulosa-luteal cell cultures treated with IFN demonstrated a significant reduction in progesterone production. Treatment with IFN/TNF, TNF, and IL-1 also decreased progesterone secretion, although statistical significance was not achieved. These findings provide evidence that a stress response occurs in human granulosa-luteal cells in response to heat and cytokines. The inhibition of gonadotrophin-sensitive steroidogenesis coincident with the induction of HSP-70 synthesis by physiological agents which are present in the corpus luteum implicates HSP-70 as a potential mediator of luteolysis in the human.