Metallothionein gene expression is regulated by serum factors and activators of protein kinase C.

Abstract

The exact physiological role of metallothionein (MT) is not clear. It has been suggested that these low-molecular-weight, highly inducible, heavy-metal-binding proteins serve in the regulation of intracellular Zn metabolism. Among the Zn-requiring systems are several enzymes involved in DNA replication and repair. Therefore, during periods of active DNA synthesis there is likely to be an increased demand for Zn, which could be met by elevated MT synthesis. For that reason, we examined whether stimulation of cellular proliferation leads to increased expression of MT. We report here that treatment of cultured mammalian cells with serum growth factors and activators of protein kinase C, all of which are known to have growth stimulatory activity, led to induction of MT mRNA. One of the required steps in the signal transduction pathways triggered by these agents, ending in MT induction, appears to be the activation of protein kinase C.Zinc (Zn) is an important component of many enzymes, including alkaline phosphatases, carboxypeptidases, dehydrogenases, RNA and DNA polymerases, and the protein synthesis machinery (see references 28 and 34 for reviews). Zn deficiency leads to severe growth retardation, developmental abnormalities, immunodeficiency, eczematoid dermatitis, and several other disorders (28). As an important ion, the homeostasis of Zn is finely maintained. Metallothioneins (MTs), which are low-molecular-weight heavy-metal-binding proteins (see reference 12 for a review), are likely to occupy a central role in the regulation of zinc metabolism (7,29). Several Zn-requiring apoenzymes can be reactivated by the transfer of Zn from MT to the apoenzyme (33). Therefore, MTs may constitute a regulatory system whose function is analogous to that of calmodulin in calcium metabolism. In addition, MTs probably serve as an intracellular reservoir of Zn. In support of these roles it has been reported that induction of MT synthesis is responsible for the increased cellular uptake of Zn after treatment with glucocorticoid hormones (13,15,16).As the major Zn-binding proteins in the cell (16), MTs can potentially contribute to many important biological processes that involve Zn-requiring enzymes, including replication, repair, transcription, protein synthesis and turnover, and energy metabolism. It is expected that during periods of active cell growth and proliferation there is an increased demand for Zn that could be met by elevated MT synthesis. In this report we describe the relationship between cellular proliferation and MT synthesis. More specifically, we examined the effect of serum and various growth factors as well as direct activators of protein kinase C, all of which are stimulators of cellular proliferation (25, 26), on the expression of MT mRNA in various mammalian cell cultures. Although MT synthesis is not directly coupled to the cell cycle, it is stimulated in response to treatment with serum, peptide growth factors, and two different activators of protein kinase C. MATERIALS AND METHODSChemicals. 12-0-Tetra...

Metallothionein RNA levels in HL‐60 cells Effect of cadmium, differentiation, and protein kinase C activation

“…There was no significant change in basal MT RNA levels upon terminal differentiation during the time frame of cell-cycle exit (6 h for differentiation with TPA [15], 1 day for differentiation with calcitriol [14], and 3-4 days with RA [17]). Thus, changes in MT RNA levels are not an early event in terminal differentiation of HL-60 cells, which argues against a central role for MT in control of cell proliferation [4,10]. However, long-term culture of cells differentiated with TPA showed a significant decrease in basal MT RNA levels (table 1).…”

“…We observed a similar, negligible effect of TPA on HepG2 cells grown under normal culture conditions (1.3-fold induction after 8 h exposure with 170 nM TPA). It is noteworthy that in fibroblasts, HeLa and HepG2 ceils, Imbra and Karin [10] reported MT induction with TPA and other mitogens after serum starvation for 40 h. MT induction following mitogenic stimulation of serumstarved cells may involve mechanisms other than, or in addition to PKC activation. It is also possible that MT induction by PKC activators is tissuespecific, much like the glucocorticoid response [2,6].…”

“…Thus, PKC activity does not correlate with, or appear necessary for, Cd 2+ induction of MT RNA. To test whether PKC activation alone is sufficient for MT induction in HL-60 cells, as suggested for serum-starved primary human fibroblasts, HeLa and HepG2 cell lines [10], both undifferen-…”

“…Though the mechanism of regulation is poorly understood, rapid induction occurs upon exposure to metals [5] and, in mammalian cells, to various circulating factors such as glucocorticoid hormones [6], ainterferon [7], and interleukin-1 and other mediators of the inflammatory response [8,9]. It has been suggested that a common pathway for MT induction involves protein kinase C (PKC) activation since MT levels increase in serum-starved cells after treatment with growth factors and TPA, agents which function in part through PKC activation [10,11]. However, data showing that multiple cis-and trans-acting factors involved in the TPA response act in a gene-and tissue-specific manner are accumulating [12].…”

Cloning and functional analysis of the hematopoietic cell‐specific phospholipase Cγ2 promoter

“…A major RNA cap site was mapped by primer extension downstream of the GC-rich stretch with several consensus sites for the transcription factor SPl, including SPl sites, which partly overlapped the consensus sites for the GC factor, reported to repress transcriptional activation of the EGF receptor gene [22]. Proximal to that cluster, an AP2 site, previously described as responsive to CAMP and TPA [23,24], was located. The promoter proximal part conferred maximal activity in a transient transfection assay.…”