Heat-induced proteolysis of HSF causes premature deactivation of the heat shock response in Nb2 lymphoma cells.

Nb2-11 cells, a prolactin (PRL)-dependent T-lymphoma cell line, display an unusual response to heat stress characterized by the lack of expression of inducible hsp70 mRNA transcripts and a reduction in the levels of constitutively expressed heat shock protein (HSP) genes. This aberrant heat shock response appears to result from heat-induced proteolytic fragmentation of heat shock factor (HSF). In this report, we have investigated processes that promote HSF fragmentation and identified characteristics of a protease that may be responsible for this effect. Cycloheximide did not affect HSF fragmentation of heat-shocked Nb2-11 cells suggesting that proteases responsible for this proteolysis are constitutively expressed and become activated by the heat shock conditions. PRL protected Nb2-11 cells from heat-induced fragmentation whereas sodium butyrate (NaBT) rendered a fragmentation-resistant cell line (Nb2-SFJCD1 cells) sensitive to HSF proteolysis. Heat-induced HSF fragmentation in Nb2-11 cells was not affected by pretreating cultures with several serine protease inhibitors. However, a dose-dependent decrease in HSF fragmentation was achieved by pretreating cultures with iodoacetamide, a cysteine protease inhibitor that is active in apoptosis. Apparently, the heat shock response in Nb2 cells is attenuated by a mechanism that involves the premature deactivation of HSF by its selective proteolysis. Attenuation of this critical cellular stress response may be an important contributor to the progression of hormone-dependent tumors possibly by influencing apoptotic processes known to regulate the activity of these cells.

Cold-induced heat shock protein expression in rat aorta and brown adipose tissue.

Recent reports indicate that Heat Shock Proteins (HSPs) are induced in mammalian tissues as part of a homeostatic response to environmental stressors. Administration of sympathomimetic drugs and neuroendocrine stress hormones has been shown to evoke an HSP response in unstressed animals indicating that cell signaling events exists that couple specific neurotransmitter/hormone-receptor interactions with HSP expression in mammalian tissues. Herein, we demonstrate that exposure of rats to a cold ambient temperature (6 degrees C) results in increased expression of constitutive and inducible members of the HSP70 gene family in association with increased expression of the mitochondrial uncoupling protein in brown adipose tissue (BAT). Increased HSP70 expression was not restricted to BAT because HSP70 was also induced in the aorta. This cold-induced HSP response is characterized by a transient increase in HSP70 protein and mRNA in both tissues during continued exposure. Ganglionic blockade prevented cold-induced HSP70 expression in BAT and aorta, indicating that sympathetic activity is requisite to this response. Administration of the alpha 1-adrenergic receptor antagonist, prazosin, also blocked expression, further delineating possible signaling mechanisms mediating this response. Apparently, cells in some mammalian tissues have adopted unique cellular regulatory mechanisms to support HSP induction that have been incorporated into the physiological response of the entire organism to an environmental stressor.

Adrenergic regulation of the heat shock response in brown adipose tissue.

One of several ways that cells respond to damage or stress is by the expression of a set of highly conserved proteins termed, heat shock proteins (HSP). Induction of the heat shock response has been positively correlated with adaptation or protection of cells and tissues from the destructive effects of various types of stressors. Although heat can induce a generalized HSP response in most cells, the selective induction of HSP in specific cell populations by pharmacological agents may prove therapeutically useful for the protection of organs or tissues at risk for damage. Results from our studies suggest that the HSP response is integrated with fundamental physiological stress responses and demonstrate that distinct regulatory events couple neurotransmitter/hormone-receptor interactions with HSP expression in mammalian tissues. We demonstrate that the adrenergic receptor agonist, phenylephrine, induces HSP expression in brown adipose tissue (BAT). Apparently, this response is mediated by alpha-adrenergic receptors in BAT because prazosin, but not propranolol, blocks HSP induction and hexamethonium is without effect. Based on the transcripts induced and the magnitude of heat shock element-binding activity, phenylephrine appears to induce HSP expression through unique transcriptional regulatory mechanisms. The phenylephrine-induced HSP response is not unique to BAT as we have found that HSP are induced in other tissues as well. In BAT, HSP may facilitate the thermogenic function of this tissue, however, their function in other tissues remains unclear. The results of this study characterize a model system where the heat shock response is differentially evoked by a specific pharmacological agent and may aide in the development of treatment strategies to selectively target HSP expression in vivo.

Thermoregulatory and heat-shock protein response deficits in cold-exposed diabetic mice.

Cold-induced expression of heat-shock proteins (HSPs) has been suggested to facilitate thermogenesis in brown adipose tissue (BAT). However, the regulation of this response and the mechanism supporting this facilitation have not been established. Because of the significant role of insulin in maintaining BAT thermogenesis, we employed a transgenic mouse model of diabetes to investigate the regulation and function of HSPs in BAT thermogenesis. These transgenic mice overexpress a calmodulin minigene regulated by the rat insulin II promotor, resulting in severe diabetes characterized by elevated blood glucose and glucagon that coincides with reduced serum and pancreatic insulin. Body temperature (Tb) of diabetic mice dropped significantly faster during a 3-h cold exposure (6 degrees C) than Tb of similarly treated control littermates. Cold exposure resulted in increased levels of constitutive and inducible HSP70 transcripts in control mice, but only constitutive HSP70 mRNA transcripts were induced in diabetic mice. Diabetes did not affect uncoupling protein induction, but cold-induced expression of members of other HSP families was reduced. Correspondingly, heat-shock regulatory factors were not activated in diabetic mice even though these factors were present. Phenylephrine induced HSP70 expression in control and diabetic animals, indicating that alpha-receptor-coupled HSP induction remained intact in BAT of diabetic mice. Insulin replacement restored the Tb response of diabetic mice as well as the HSP response. From these results it is clear that physiological signals that regulate cold-induced activation of BAT also regulate HSP expression in this tissue. This diabetic model provides a novel system in which the HSP response to cold has been selectively knocked out, making it a useful tool for the study of HSP regulation and function in BAT.

A novel heat shock response in prolactin-dependent Nb2 node lymphoma cells.

Virtually all cells respond to heat stress by increased expression or induction of one or more of the highly conserved cellular stress response proteins, heat shock proteins (HSPs). Here, we report the unusual property of rat Nb2-11 cells, a prolactin-dependent pre-T-cell line, to display reduced HSP expression following exposure to elevated temperature. After heat stress (41 degrees C, 1 h), there was no evidence of inducible members of the 70 kDa HSP family, a response common to other cell culture and tissue systems. Moreover, expression of constitutive members of the HSP70 and HSP90 families decreased during the heat stress, apparently reflecting a decrease in mRNA stability. Gel shift assays revealed that heat shock factor (HSF) was activated in spite of the lack of expression of inducible HSP70 transcripts, although its DNA binding rapidly deteriorated. Immunoblotting, using an antibody specific to HSF1, indicated that proteolysis of HSF1 may be responsible for this rapid termination of heat shock element binding. CCAAT binding, a component of constitutive HSP70 expression, was also reduced by heat stress in Nb2-11 cells and may account for the decline in constitutive HSP70 expression. Prolactin pretreatment prevented the fragmentation of HSF1, protected heat shock element and CCAAT binding, prevented the decline in constitutive HSP70 and HSP90 expression, and restored a modest expression of inducible HSP70 following heat treatment. Results of this study describe a unique regulatory defect in HSP expression in Nb2-11 cells, possibly a common characteristic of other hormone-dependent tumors.

Characterization and regulation of cold-induced heat shock protein expression in mouse brown adipose tissue.

The accumulation of heat shock proteins (HSPs) after the exposure of cells or organisms to elevated temperatures is well established. It is also known that a variety of other environmental and cellular metabolic stressors can induce HSP synthesis. However, few studies have investigated the effect of cold temperature on HSP expression. Here we report that exposure of Institute of Cancer Research (ICR) mice to cold ambient temperatures results in a tissue-selective induction of HSPs in brown adipose tissue (BAT) coincident with the induction of mitochondrial uncoupling protein synthesis. Cold-induced HSP expression is associated with enhanced binding of heat shock transcription factors to DNA, similar to that which occurs after exposure of cells or tissues to heat and other metabolic stresses. Adrenergic receptor antagonists were found to block cold-induced HSP70 expression in BAT, whereas adrenergic agonists induced BAT HSP expression in the absence of cold exposure. These findings suggest that norepinephrine, released in response to cold exposure, induces HSP expression in BAT. Norepinephrine appears to initiate transcription of HSP genes after binding to BAT adrenergic receptors through, as yet, undetermined signal transduction pathways. Thermogenesis results from an increase in activity and synthesis of several metabolic enzymes in BAT of animals exposed to cold challenge. The concomitant increase in HSPs may function to facilitate the translocation and activity of the enzymes involved in this process.

Neural and endocrine mechanisms of cocaine-induced 70-kDa heat shock protein expression in aorta and adrenal gland.

Cocaine has properties of a physiologic stressor that are reflected by its ability to activate both the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. We have previously reported that activation of the hypothalamic-pituitary-adrenal axis and sympathetic nervous system by restraint and pharmacologic agents induces expression of a set of highly conserved cellular stress response proteins (heat shock proteins, HSP) in the adrenal gland and aorta. In the adrenal gland, HSP expression appears to be mediated by stress-induced increases in adrenocorticotropic hormone whereas expression in the aorta involves noradrenergic neurotransmission. In this report we capitalize on the ability of cocaine to stimulate physiologic stress responses to define further mechanisms regulating HSP70 expression in these tissues. We report the novel observation that cocaine administration induces both adrenal and vascular HSP70 mRNA expression. Elevated HSP70 mRNA was preceded by activation of factors capable of binding to the heat shock transcriptional control element and was followed by an elevation in HSP70 protein. Cocaine significantly increased plasma adrenocorticotrophic hormone whereas hypophysectomy eliminated cocaine-induced expression in the adrenal gland suggesting that in this tissue, the effect of cocaine on HSP70 expression is also mediated via adrenocorticotrophic hormone. In the aorta, depletion of catecholamines by reserpine pretreatment paradoxically augmented cocaine-induced HSP70 expression. Based on these results, it appears that HSP70 expression in the aorta occurs through direct actions of cocaine on vascular cells that are ultimately transduced to activation of the HSP70 gene rather than indirectly through alterations in catecholamine reuptake and release.(ABSTRACT TRUNCATED AT 250 WORDS)