Progress 11/01/00 to 07/31/04
Outputs
We have shown previously that removal of zinc with a chelator, DTPA, enhances the induction of growth hormone gene expression by thyroid hormone in rat pituitary tumor cells. The aim of this proposal was to determine whether other thyroid hormone target genes in other cell types respond in this unexpected way to lack of zinc and to investigate the mechanisms underlying these effects. In freshly isolated rat pituitary cells, growth hormone gene expression was not responsive to either thyroid hormone or DTPA. However, TSH expression was inhibited, as expected, by thyroid hormone, whereas DTPA stimulated TSH expression both in the absence and presence of thyroid hormone. This stimulation was inhibited by zinc. Thus freshly isolated pituitary cells do not respond in the same way to either thyroid hormone or DTPA as do pituitary tumor cells, but altering zinc availability does impact TSH responsiveness. We have also investigated the effects of zinc removal in cultured
primary rat hepatocytes. Addition of chelator amplified the effects of thyroid hormone on gene expression, and this effect was reversed by addition of zinc. Furthermore, zinc by itself inhibited T3-induced gene expression in a dose-dependent manner. These effects were mRNA specific, since the expression of constitutively expressed genes was not affected. GH3 cells and primary hepatocytes differ in their handling of zinc, but the negative association between T3 action and zinc availability is quite consistent. Zinc-65 was used to test the effects of DTPA on cellular zinc flux. Uptake of Zn-65 by both hepatocytes and pituitary tumor cells was greatly reduced by DTPA. When hepatocytes were prelabeled with Zn-65, DTPA also increased efflux of zinc. However, when GH3 cells were preloaded with Zn-65, addition of DTPA to the media actually increased label retention by the cells. Kinetic experiments with GH3 cells showed that DTPA results in a rapid blockage of Zn-65 efflux from the cells.
Thus the activity of existing transporters appears to be affected. These experiments were extended to H4IIE hepatoma cells. These cells responded to DTPA by reducing zinc efflux, a response similar to GH3 cells but different from primary hepatocytes. To investigate the mechanism whereby DTPA enhanced the T3 responsiveness of GH mRNA, a construct containing the GH promoter ligated to a luciferase reporter gene was transfected into GH3 cells by electroporation. Surprisingly, DTPA decreased the activity of this promoter, whether or not T3 was present, in a manner reversible by zinc. Suspecting that the transgene failed to mimic the endogenous promoter because of its episomal organization in the nucleus, stable transfections were also performed. In these cells, treatment with combinations of T3/DTPA/Zn revealed a pattern of expression very similar to the endogenous GH mRNA. This confirmed that DTPA functioned through the GH promoter to increase transcription but that stable incorporation
into the chromatin environment was required.
Impacts
Alterations in zinc availability affects many nuclear processes, but, for the most part, the underlying mechanisms are unknown. These studies have shown unexpected effects of zinc deficiency, in that thyroid hormone action was actually enhanced at the transcriptional level. Further understanding these mechanisms is important for what it reveals about both thyroid hormone and zinc. The fact that some cultured cells respond to a reduction in available zinc by shutting down efflux may well reflect the situation seen in humans and animals where zinc concentrations are maintained in selected tissues in the face of deficiency. Given that the two cell lines in which this behavior was observed were both derived from tumors, there is also the intriguing possibility that maintaining cellular zinc is a crucial adaptation required for the enhanced growth needs of these cells.
Publications
- Sciaudone, M.P., Yao, L., Schaller, M., Zinn, S.A., Freake, H.C. 2004. Diethylenetriaminepentaacetic acid enhances thyroid hormone action by a transcriptional mechanism. Biological Trace Element Research. 99:219-231.
- Kang, H.W., Freake, H.C. 2004. Zinc inhibits the induction of S14 mRNA by T3 in primary rat hepatocytes. FASEB J 18:A102.
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Progress 01/01/03 to 12/31/03
Outputs
We have shown previously that removal of zinc with a chelator, DTPA, enhances the induction of growth hormone (GH) gene expression by thyroid hormone (T3) in GH3 rat pituitary tumor cells. The aim of this proposal is to determine whether other thyroid hormone target genes in other cell types respond in this unexpected way to lack of zinc and to investigate the mechanisms underlying these effects. In primary rat hepatocytes, addition of DTPA amplified the effects of T3 on mRNA S14 gene expression, and this effect was reversed by addition of zinc. However, differently from GH3 cells, zinc by itself inhibited T3-induced gene expression in a dose-dependent manner. These effects were mRNA specific, since the expression of constitutively expressed genes was not affected. However, they were not specific to zinc. Dose-response experiments with other divalent cations demonstrated a range of effects on S14 mRNA. Cobalt, nickel and manganese produced inhibitory effects similar
to zinc. Copper was toxic to the cells at 40 uM and magnesium was without effect. Thus while both primary hepatocytes and pituitary tumor cells respond to T3/DTPA/zinc treatments, these responses do vary. Different responses to DTPA have also been noted between these two cell types using a zinc radio-isotope, Zn-65. We had previously showed that addition of DTPA to the medium blocked uptake of Zn-65 both in hepatocytes and GH3 cells. However, when cells were pre-labeled, while DTPA increased transfer of Zn-65 from hepatocytes to media, it resulted in greater retention of tracer by GH3 cells. These observations were followed up with time course studies of Zn-65 efflux from pre-labeled cells. In GH3 cells exposed to DTPA, while there was a rapid loss of presumably loosely associated isotope from the cells, cellular Zn-65 concentrations were thereafter maintained for the ensuing 6 hours. In the absence of DTPA, a continuous efflux of Zn-65 was observed. In hepatocytes, Zn-65 levels
dropped continuously over time and the rate of loss was greater in the presence of DTPA than in its absence. We hypothesize that these cell-specific responses reflect differences in expression or regulation of zinc transporters. The mechanism underlying the stimulatory effects of DTPA in GH3 cells has been probed by transfection of a 530 bp GH promoter/luciferase reporter construct. We had found in transient transfections, that DTPA inhibited the activity of this construct, both in the presence and absence of T3. This response was quite different from that seen with the endogenous GH mRNA. We therefore prepared GH3 cell lines stably transfected with the same construct. Treatment of these cells with T3/DTPA/zinc produced parallel responses between luciferase activity and endogenous GH mRNA, namely an enhancement of T3-stimulation by DTPA that was reversed by zinc. DTPA had no effects in the absence of T3. Thus DTPA is working at the level of transcription in GH3 cells, but gene
incorporation into chromatin appears to be necessary for these effects to be seen. We therefore suggest that nuclear coregulatory proteins are the site of zinc T3 interaction within the nucleus.
Impacts
Alterations in zinc availability affects many nuclear processes, but, for the most part, the underlying mechanisms are unknown. These studies will delineate the interaction of zinc with a nuclear acting hormone, thyroid hormone and thus clarify some of these mechanisms. Zinc can often be suboptimal in the diet, particularly when energy is limited, and determining these mechanisms may be important for understanding the symptoms of those deficiencies.
Publications
- Sciaudone MP, Yao L, Schaller M, Zinn SA and Freake HC. 2004. Diethylenetriaminepentaacetic acid enhances thyroid hormone action by a transcriptional mechanism. Biological Trace Element Research. In press.
- Yao L, Sciaudone MP, Zinn SA and and Freake HC. 2003. Chelation of zinc enhances thyroid hormone activation of a stably transfected growth hormone promoter. FASEB J 17, A1159.
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Progress 01/01/02 to 12/31/02
Outputs
We have shown previously that removal of zinc with a chelator, DTPA, enhances the induction of growth hormone gene expression by thyroid hormone in rat pituitary tumor cells. The aim of this proposal is to determine whether other thyroid hormone target genes in other cell types respond in this unexpected way to lack of zinc and to investigate the mechanisms underlying these effects. In freshly isolated rat pituitary cells, growth hormone gene expression was not responsive to either thyroid hormone or DTPA. The reason for this lack of responsiveness is unclear. However, TSH expression was inhibited, as expected, by thyroid hormone, whereas DTPA stimulated TSH expression both in the absence and presence of thyroid hormone. This stimulation was inhibited by zinc. Thus freshly isolated pituitary cells do not respond in the same way to either thyroid hormone or DTPA as do pituitary tumor cells, but altering zinc availability does impact TSH responsiveness. We have also
investigated the effects of zinc removal in cultured primary rat hepatocytes. Addition of chelator amplified the effects of thyroid hormone on gene expression, and this effect was reversed by addition of zinc. Furthermore, zinc by itself inhibited T3-induced gene expression in a dose-dependent manner. These effects were mRNA specific, since the expression of constitutively expressed genes was not affected. However, they were not specific to zinc, since inhibition of T3-induced gene expression was observed with some other divalent cations. This extends the findings of zinc:T3 interaction beyond the pituitary into an organ of great metabolic significance. A zinc radio-isotope, Zn-65, was used to test the effects of the chelator on cellular zinc movement. When the culture media was labeled with Zn-65, accumulation of radioactivity by both hepatocytes and pituitary tumor cells was greatly reduced by DTPA, suggesting that the chelator bound zinc in the media and decreased its uptake. When
hepatocytes were preloaded with Zn-65, DTPA also increased efflux of zinc. However, when GH3 cells were preloaded with Zn-65, addition of DTPA to the media actually increased label retention by the cells. This response occurred rapidly after addition of DTPA and appeared to be due to an inhibition of efflux of zinc from the cells. These differential findings in pituitary cells and hepatocytes may reflect the situation in vivo where tissues vary in the extent to which they give up zinc in response to deficiency. The mechanism underlying the stimulatory effects of DTPA in GH3 cells has been probed by transfection of a GH promoter/luciferase reporter construct. In transient transfections, DTPA inhibited the activity of this construct, both in the presence and absence of T3. This response was quite different from that seen with the endogenous GH mRNA. However, when stably transfected cells were prepared, luciferase activity was stimulated by DTPA and only in presence of T3. Thus,
incorporation of the transgene into the chromatin environment appears to be required for its response to DTPA.
Impacts
Both zinc and thyroid hormone exert profound but similar effects on growth and metabolism though exactly how is not clear, particularly for zinc. This project will improve understanding of the mechanisms by which these important regulators work and determine the extent to which they are interrelated.
Publications
- Freake HC, Schaller M, Trzcienski A and Zinn SA. 2002. Zinc chelation amplifies thyroid hormone action, but has variable effects on zinc efflux in cultured cells. Proceedings of the Nutrition Society 61, 47A.
- Schaller MA, Trzcienski AL and Freake HC. 2002. The influence of diethylenmetriaminepenta acetic acid on cellular zinc movement. FASEB J 16, A975.
- Trzcienski AL and Freake HC. 2002. Chelation of zinc enhances induction of S14 mRNA in primary rat hepatocytes. FASEB J 16, A232.
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Progress 01/01/01 to 12/31/01
Outputs
Both zinc and thyroid hormone play critical roles in support of the growth and development of humans and other animals, though the exact mechanisms are unclear. The possibility of an interaction between their effects exists because zinc is thought to be important for the structure and function of the thyroid hormone receptor, a key protein that mediates the actions of thyroid hormone in the nucleus. However, we have shown previously that removal of zinc with a chelator, DTPA, enhances the induction of growth hormone gene expression by thyroid hormone in rat pituitary tumor cells. The aim of this proposal is to determine whether other thyroid hormone target genes in other cell types respond in this unexpected way to lack of zinc and to investigate the mechanisms underlying these effects. The effects of DTPA were tested in pituitary cells freshly isolated from young rats to see whether they extended beyond tumor cells. The cell system was shown to be active and
responsive, since growth hormone gene expression was reduced by addition of somatostatin. However, in these cells growth hormone gene expression was not responsive to either thyroid hormone or DTPA. The reason for this lack of responsiveness is unclear and being investigated. However, TSH expression was inhibited, as expected, by thyroid hormone, whereas DTPA stimulated expression both in the absence and presence of thyroid hormone. This stimulation was inhibited by zinc. Thus freshly isolated pituitary cells do not respond in the same way to either thyroid hormone or DTPA as do pituitary tumor cells. Nevertheless, altering zinc availability does impact TSH responsiveness. We have also investigated the effects of zinc removal in cultured primary rat hepatocytes. In this system, addition of chelator amplified the effects of thyroid hormone on gene expression, and this effect was reversed by addition of zinc. Furthermore, zinc by itself inhibited T3-induced gene expression in a
dose-dependent manner. These effects were specific, since the expression of constitutively expressed genes was not affected. This demonstration of an interaction between thyroid hormone and zinc chelation in primary hepatocytes is important, since it extends the findings beyond the pituitary into an organ of great metabolic significance. A zinc radio-isotope, Zn-65, was used to test the effects of the chelator on cellular zinc levels. When the culture media was labeled with Zn-65, accumulation of radioactivity by both hepatocytes and pituitary tumor cells was greatly reduced by DTPA, suggesting that the chelator bound zinc in the media and decreased its uptake. When hepatocytes were preloaded with Zn-65, DTPA also increased efflux of zinc. However, when GH3 cells were preloaded with Zn-65, addition of DTPA to the media actually increased label retention by the cells. These differential findings in pituitary cells and hepatocytes may reflect the situation in vivo where tissues vary in
the extent to which they give up zinc in response to deficiency. It is unclear at this point how cells monitor their zinc status in order to initiate the different responses recorded in these experiments.
Impacts
Zinc deficiency leads to multiple deficits, including impairment of growth and compromised immune function. The underlying mechanisms are unclear and this project is using cell culture models to help define these mechanisms.
Publications
- Sciaudone, MP, Schaller MA and Freake HC. Zinc chelation inhibits the activity of the transfected growth hormone promoter in GH3 cells. FASEB J 15, A257, 2001.
- Rivard AL, Schaller MA, Freake HC and Zinn SA. The effects of zinc and thyroid hormone on the expression of growth hormone and thyroid stimulating hormone in primary rat anterior pituitary cells. J Anim Sci. 79 (Suppl 1): 435-436, 2001.
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