zinc depletion and stem cell

[Guest guest]

Effects of intracellular zinc depletion on metallothionein and ZIP2

transporter expression and apoptosis

Jay Cao, A. Bobo, P. Liuzzi and J. Cousins

Food Science and Human Nutrition Department and Center for

Nutritional Sciences, University of Florida, Gainesville 32611-0370

Correspondence: J. Cousins, Food Science and Human Nutrition

Department, University of Florida, 201 FSHN, P.O. Box 110370,

Gainesville, FL 32611-0370. E-mail: cousins@...

ABSTRACT

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ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

REFERENCES

Zinc is critical for the functional and structural integrity of

cells. We have used the monocytic cell line THP-1 as a model in which

to study both the responsiveness of metallothionein and ZIP2

transporter expression to zinc depletion induced by the intracellular

zinc chelator TPEN [N,N,N',N'-tetrakis(2-pyridylmethyl)

ethylenediamine] and the extent of concomitant apoptosis.

Metallothionein expression increased proportionately with the

addition of zinc to the medium and decreased with TPEN treatment.

When treated with TPEN, both THP-1 cells and human peripheral blood

mononuclear cells exhibited marked decreases in cellular zinc

concentrations and increases in ZIP2 mRNA expression. These results

suggest that cells attempt to homeostatically adjust to zinc

depletion. When THP-1 cells were treated with >5 µM TPEN, cell

viability decreased, and cells entered the early stages of apoptosis.

These data show that metallothionein and ZIP2 expression are

inversely related during zinc depletion and that apoptosis is

concurrent with these changes.

Key Words: monocytes • PCR • regulation

INTRODUCTION

TOP

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

REFERENCES

Zinc is critical for the functional and structural integrity of cells

and contributes to a number of important processes including gene

expression [1 2 3 ]. Pools used to supply zinc for these functions

are regulated by transporters at the plasma membrane as well as at

intracellular sites [reviewed in ref. 4 ]. Studies with intact

animals and cells have delineated a scenario of regulation including

glucocorticoid hormones and those hormones mediated via cAMP- and

cytokine-induced changes [1 ]. At the hepatic level, the

glucocorticoid, insulin and glucagon produce transient dysregulation

of zinc metabolism, which produces a decrease in plasma zinc

concentrations. Similarly, immune-regulatory peptides, including

interleukins 1 and 6, produce tissue-specific changes in zinc

metabolism [1 ]. The liver is also a key component of this metabolic

response to infection and oxidative stress. Expression of

metallothionein (MT), a cysteine-rich zinc-binding protein, appears

to be linked to these metabolic changes [5 6 ]. Much less is known

about zinc metabolism and function in rapidly growing cells,

including reticulocytes and stem cell precursors of leukocytes, or

how MT and/or zinc transporters regulate zinc metabolism and function

in such cells.

Our experiments with human subjects have shown that MT expression is

altered when the dietary zinc supply is restricted or supplemented.

Erythrocyte MT protein concentrations, as measured by enzyme-linked

immunosorbent assay (ELISA), are reduced or elevated, after a lag

period of 6 days, when the dietary zinc intake of these subjects is

correspondingly adjusted [7 8 ]. Similar changes have been observed

in red blood cells from zinc-deficient rats [9 ]. MT protein

concentrations in human leukocyte populations are lower than those in

red blood cells [10 ]; however, MT mRNA levels can be measured by

competitive reverse transcriptase (RT)-PCR [8 11 ]. This approach has

allowed direct measurement of MT mRNA abundance in purified monocytes

(the type of leukocyte that has the highest MT expression), as well

as in peripheral blood mononuclear cells (PBMCs) and in leukocytes on

dried blood spots obtained from zinc-supplemented subjects [11 ]. MT

mRNA levels are quite sensitive to increases in zinc supplementation,

suggesting that leukocytes, particularly monocytes, are an attractive

model in which to examine zinc function. This interest is enhanced by

observations that zinc alters the susceptibility of cells to

apoptosis [12 ], which may relate to a key function of this

micronutrient.

We have been using THP-1 cells, a human monocytic cell line, as a

model for studying zinc metabolism and function in immune cells

[13 ]. One goal of our experiments is to develop a method that allows

the use of leukocytes for assessing dietary zinc status in

populations. There is evidence to suggest that marginal zinc

deficiency, which has no recognized laboratory method for

quantitation, is more widespread than previously believed and

produces morbidity worldwide [14 15 ]. As has been shown previously

[8 11 ], induction of MT mRNA expression in monocytes is influenced

by the zinc supply. Furthermore, recent evidence has shown that zinc

transporter expression in rat intestine, liver, and kidney is also

zinc dependent [16 ]. Comparable information on leukocyte zinc

transporters has not been obtained. Consequently, a second goal of

the current experiments with THP-1 cells is to examine the

responsiveness of the zinc transporter ZIP2 to decreased zinc levels.

ZIP2 is a member of the ZIP (ZRT1, IRT1-like) family of proteins.

Data from transfection studies with human cells strongly suggest that

ZIP2 is an importer and that it is zinc regulated [17 ].

The purposes of the present studies were (1) to examine in both THP-1

cells and human PBMCs the effects of intracellular zinc depletion

induced by a zinc chelator on MT and ZIP2 expression, the extent of

apoptosis as a function of zinc depletion, and the relationship of MT

and ZIP2 expression to apoptosis and (2) to correlate intracellular

zinc levels, using a new cell-permeating zinc probe, with the

measurable changes in MT and ZIP2 levels and apoptosis.

Regulation of the Saccharomyces cerevisiae EKI1-encoded Ethanolamine

Kinase by Zinc Depletion*

C. Kersting and M. Carman1

From the Department of Food Science, Cook College, New Jersey

Agricultural Experiment Station, Rutgers University, New Brunswick,

New Jersey 08901

Received for publication, February 21, 2006 , and in revised form,

March 15, 2006.

ABSTRACT

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ABSTRACT

INTRODUCTION

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

REFERENCES

Ethanolamine kinase catalyzes the committed step in the synthesis of

phosphatidylethanolamine via the CDP-ethanolamine branch of the

Kennedy pathway. Regulation of the EKI1-encoded ethanolamine kinase

by the essential nutrient zinc was examined in Saccharomyces

cerevisiae. The level of ethanolamine kinase activity increased when

zinc was depleted from the growth medium. This regulation correlated

with increases in the CDP-ethanolamine pathway intermediates

phosphoethanolamine and CDP-ethanolamine, and an increase in the

methylated derivative of phosphatidylethanolamine,

phosphatidylcholine. The -galactosidase activity driven by the PEKI1-

lacZ reporter gene was elevated in zinc-depleted cells, indicating

that the increase in ethanolamine kinase activity was attributed to a

transcriptional mechanism. The expression level of PEKI1-lacZ

reporter gene activity in the zrt1zrt2 mutant (defective in plasma

membrane zinc transport) cells grown with zinc was similar to the

activity expressed in wild-type cells grown without zinc. This

indicated that EKI1 expression was sensitive to intracellular zinc.

The zinc-mediated regulation of EKI1 expression was attenuated in the

zap1 mutant defective in the zinc-regulated transcription factor

Zap1p. Direct interactions between Zap1p and putative zinc-responsive

elements in the EKI1 promoter were demonstrated by electrophoretic

mobility shift assays. Mutations of these elements to a nonconsensus

sequence abolished Zap1p-DNA interactions. Taken together, this work

demonstrated that the zinc-mediated regulation of ethanolamine kinase

and the synthesis of phospholipids via the CDP-ethanolamine branch of

the Kennedy pathway were controlled in part by Zap1p.

Zinc depletion happens as result of folate and iron. Folate

depletes zinc and iron competes for zinc[think prenatal vits] Then

comes the hep-b at birth with the toxins and saccharomyces

cerevisiae! Also zinc depletion affects the Mt.