The membrane, magnesium, mitosis (MMM) model of cell proliferation control

ARTICLE

Auteur(s) : Harry Rubin

Department of Molecular and Cell Biology, Life Sciences Addition, University of California, Berkeley, CA 94720-3200, USA

Physiology of growth stimulation

The rate of DNA synthesis of course varies coordinately with the rate of cell proliferation. The initiation of DNA synthesis can be brought to a very low rate by sharply lowering the serum concentration overnight in confluent cultures. The restoration of serum to its original high level in such inhibited cultures is followed by a lag of several hours before DNA synthesis begins to increase, and it rises to a maximum in 15-20 hr. Several other physiological processes which are inhibited by confluence and withdrawal of serum are very rapidly stimulated by the restoration of serum. These processes include the uptake of glucose and its utilization, the phosphorylation of uridine, and most importantly, the synthesis of protein. The increase in glucose uptake is not essential for a single round of accelerated DNA synthesis, and the uptake and phosphorylation of uridine is totally gratuitous since there is ordinarily no uridine in the medium. In contrast, the onset of DNA synthesis is acutely dependent on the rate of synthesis and accumulation of protein, particularly of ribosomal protein. The combination of these effects is known as the coordinate response [3].

The stimulation provided by added serum must be continuously maintained for the entire lag period to initiate an increase in the rate of DNA synthesis [4]. In fact, acute inhibition of protein synthesis by cycloheximide during the rise of DNA synthesis stops any further increase in its rate [5]. Inhibition of RNA synthesis by actinomycin D has a delayed effect on DNA synthesis, probably by exhausting the supply of messenger RNA. The increase in protein synthesis which occurs promptly upon the addition of serum is dependent on the rate of the initiation step rather than the rate of chain elongation. Although serum or a combination of the purified polypeptide growth factors present in serum is necessary to maintain the continuous proliferation of cells, the early responses to such physiological agents as well as the later onset of DNA synthesis can be stimulated by non-specific treatments. For example, confluent chicken embryo fibroblasts can be stimulated in this manner by subtoxic concentrations of heavier metals such as zinc, cadmium, manganese, mercury and lead. Trypsin and other proteolytic enzymes, in amounts too small to cause retraction much less detachment of cells, also stimulate chicken embryo fibroblasts. Such treatments are not able to stimulate established lines of mouse fibroblasts, but sodium pyrophosphate at a sharply defined low concentration, just sufficient to form floccular precipitates with calcium in the medium, is a potent stimulator of the coordinate response [6, 7].

The accumulated evidence indicates that all the stimulatory agents, whether physiological, and therefore specific, or non-physiological and not specific, initiate the coordinate response by direct action on the plasma membrane. The restriction of action to the plasma membrane is made particularly evident in the case of pyrophosphate because of the strict dependence of stimulation on the formation of insoluble floccules, which appear suddenly at a fixed concentration that is dependent on the concentration of calcium and inorganic phosphate in the medium [7]. If the floccules are prevented from combining with the plasma membrane by completely filling a closed culture dish with medium and inverting it, the floccules sink to the bottom and there is neither contact with the cells nor stimulation [6]. A further indicator of a central role for membrane activity in regulating the coordinate response is the requirement for growth for normal cells to attach to and spread on a solid substratum. If the cells are kept in a spherical shape in suspension, or, are squeezed together in a crowded culture, they will not proliferate.

Role of Mg²⁺ in regulating the coordinate response of cells to growth factors and non-specific stimulators

The experimental strategy in examining the role of Mg²⁺ in growth regulation was to reduce its concentration in the medium and observe the effect on various components of the coordinate response. The initial results were erratic, since a drastic reduction in Mg²⁺ concentration was required to achieve inhibition. As it turned out, it was difficult to get the cells to give up their Mg²⁺, not surprisingly given the essential nature of Mg²⁺ for so many cellular activities. The use of EDTA to chelate Mg²⁺ was no help since it has a much stronger preference for Ca²⁺. However, pyrophosphate came to our aid because it binds Mg²⁺ somewhat more than Ca²⁺. Although very small amounts of pyrophosphate stimulate the proliferation of Balb 3T3 fibroblasts in an excess of Mg²⁺, when the pyrophosphate concentration exceeds that of Mg²⁺ there is a reproducible reduction in synthesis of protein, RNA and DNA.

It was later found that deprivation of Ca²⁺ in confluent cultures of mouse fibroblasts inhibited DNA synthesis, but the effect could be reversed with excess Mg²⁺[8]. In contrast, inhibitory effects resulting from deprivation of Mg²⁺ could not be reversed by raising the Ca²⁺ concentration of the medium, indicating that Mg²⁺ is the operational element. These experiments showed that deprivation of Ca²⁺ concentration in the medium facilitated the manipulation of the Mg²⁺ concentration in the cells over a wide range, as a function of the external Mg²⁺ concentration. There is an optimal concentration of Mg²⁺ for protein and DNA synthesis beyond which there is a sharp decrease in both processes [9]. The resulting bell-shaped curve for protein and consequently DNA synthesis as a function of Mg²⁺ resembled the curve produced for cell-free synthesis of protein by a purified preparation of ribosomes [10]. Change in the rate of protein synthesis is extremely sensitive to any change in the concentration of Mg²⁺, up or down, indicating that the free Mg²⁺ of the cell is normally set at a concentration that would alter the rate of protein synthesis with any change in free Mg²⁺, no matter how small. The Mg²⁺-dependent change in protein synthesis precedes any change in DNA synthesis by hours, consistent with the key regulatory roles of Mg²⁺ and protein synthesis in cell proliferation [11]. The cell responds to a moderately inhibitory increase of intracellular Mg²⁺ concentration by actively reducing its concentration to normal levels over a period of hours, with a consequent increase in protein synthesis and a delayed increase in DNA synthesis, indicating how crucial Mg²⁺ is to the cellular growth economy. It is notable that there is no direct effect of Mg²⁺ concentration on the rate of DNA synthesis. If the Mg²⁺ concentration is raised to extremely high concentrations, the cell cannot lower its internal Mg²⁺, and neither protein nor DNA synthesis recovers, leading to death of the cells.

While protein synthesis is the crucial reaction to the Mg²⁺ levels in cells that determine DNA synthesis and proliferation, the effects are probably mediated through protein kinase cascades. Insulin acts as a growth factor by combining with a plasma membrane receptor and activating a tyrosine kinase on the cytoplasmic side of the membrane [1, 2]. The tyrosine kinase in turn activates the PI 3-K cascade consisting mainly of serine/threonine kinases combined with inhibitory proteins which lead to phosphorylation of the mTOR kinase. The mTOR kinase in turn phosphorylates two proteins involved in the initiation of translation. Increased synthesis of ribosomal proteins and elongation factors are especially important in accumulating protein and initiating DNA synthesis. While most of the protein kinases in this pathway have a very low Km for MgATP^2-, mTOR has a high requirement that approximates the level of free Mg²⁺ in the cell. The MMM model holds that any rise in free Mg²⁺ increases MgATP^2- and activates mTOR, thereby accelerating the initiation of translation, and speeding the passage of cells through the G1 period to DNA synthesis and mitosis.

Direct evidence for the increase of free Mg²⁺ in growth stimulation of cells

Subsequently a Mg²⁺-sensitive indicator, mag-fura-2, was developed [18, 19] which could be used to measure intracellular free Mg²⁺ stimulated by growth factors. Epidermal growth factor increased DNA synthesis 48-fold in a serum-starved line of muscle cells [20]. Free Mg²⁺ increased after a 5 min lag period from an initial 0.32 mM to as high as 1.4 mM at 20 min, and then leveled off. Swiss 3T3 cells stimulated by insulin increased free Mg²⁺ in 30-60 min from a basal level of 0.22 mM to a final value of 0.29-0.35 mM [21]. Estimates of the free Mg²⁺ level were unreliable beyond 1 hr because of mag-fura-2 leakage from the cells, but the results were consistent with the long term increase in free Mg²⁺ as required to carry cells through the cell cycle. It is assumed that a significant fraction of the free Mg²⁺ was released from the internal surface of the perturbed membrane where it is bound to fixed sites, but the increase in total Mg²⁺ probably came from increased activity of Na/K ATPase pumps of Mg²⁺ in the membrane [22].

The effect of Mg²⁺ and other cations on protein synthesis could be determined in the oocytes of the frog Xenopus laevis, which are large enough to allow direct injection of the cations and measurement of protein synthesis in a single cell by incorporation of ³H-leucine [23]. Initial results indicated that injection of K⁺ into the oocytes increased the rate of protein synthesis to a similar extent as did exposure to gonadotropin, the physiological method for starting embryonic development. There were several inconsistencies in the results however that indicated that K⁺ played an indirect role in the activation of protein synthesis [24]. The injection of Mg²⁺, however, stimulated protein synthesis at a much lower concentration than K⁺, giving a sharper peak in a bell-shaped curve and exhibiting no inconsistencies. It was concluded that K⁺ (or Na⁺) displaced Mg²⁺ from bound sites and made it directly available for protein synthesis. Ion-sensitive microelectrodes were used to determine free K⁺ and Mg²⁺, and gave the usual low values for the latter. How gonadotropin sets off these changes remains to be determined but it may have to do with cation exchanges initiated by perturbation of the cell membrane as suggested for stimulation of somatic cells by growth factors [15, 16].

In contrast to the acceleration of protein synthesis as the key response for entraining cell proliferation, the increased uptake of uridine has no apparent physiological role in the process. That is because uridine is not ordinarily added in the growth medium, and its addition is devoid of effect on proliferation. However, phosphorylation of uridine by uridine kinase is the limiting step in its trapping by cells [25], a simple enzymatic reaction that requires MgATP^2- and can be studied in cells or in cell-free extracts. Deprivation of Mg²⁺ in cultures reduces the rate of uridine trapping by cells, in the same manner as does the removal of serum, i.e., by reducing the Vmax for Mg²⁺ of the reaction [26]. In contrast, the uptake of thymidine is unaffected by changing the concentration of Mg²⁺ in the medium. This difference can be explained by the fact that the Km of uridine kinase for Mg²⁺ is 10-fold higher than the Km of thymidine kinase for Mg²⁺. Hence, the phosphorylation of thymidine in cells has sufficient cellular Mg²⁺ for maximum activity even without growth factors or significant external Mg²⁺, while the phosphorylation of uridine is sensitive to both treatments. Raising the intracellular Mg²⁺ concentration high enough in the presence of the divalent cation ionophore A21387 and high extracellular Mg²⁺ increases uridine trapping to the same extent as addition of serum to the cells. Mg²⁺ also relieves feedback inhibition of uridine kinase by UTP and CTP [27]. The sum of these observations supports a regulatory role for Mg²⁺ in the gratuitous uptake of uridine, and reinforces its role in the other more essential responses to growth factors.

Discussion

The most significant early response of cells to growth factors is an increased rate of translation which is governed by the initiation step [29]. It has long been recognized that initiation of protein synthesis has a higher Mg²⁺ requirement than chain elongation [30, 31]. The rate of protein synthesis proved to be directly proportional to change in the Mg²⁺ content of cells [9, 11]. The changes in Mg²⁺ and protein synthesis had to be maintained throughout the G1 period to produce their full effect. At very high Mg²⁺ concentration there was an inhibition of protein synthesis just as there is in cell-free extracts. The sum of these observations lent strong support to a central role of Mg²⁺ in coordinating multiple biochemical responses of cells to growth factors. This support was reinforced by measurements of free Mg²⁺ levels in cells after stimulation. A particularly significant contribution to understanding the role of Mg²⁺ in activating protein synthesis came from the separate injection and measurement of the four major cellular cations into frog oocytes with observation of their effects on the initiation of protein synthesis, which left no doubt that increase in free Mg²⁺ is the direct effector of the process which begins growth and development of the frog set off physiologically by gonadotropin [24].

Given the cumulative evidence for a central role of Mg²⁺ in the coordinate response of cells to growth stimuli, it is instructive to consider why there was so much resistance to accepting the hypothesis. Possibly the most widely read review of early signals in the mitogenic response gives detailed attention to Na⁺, K⁺, Ca²⁺ and H⁺ without mention of Mg²⁺[32]. Nor does the review acknowledge the early and persistent increase in protein synthesis and its essential relation to the onset of DNA synthesis [5, 29, 33-35]. One might think that the evidence for the essential role of increased protein synthesis would call attention to its sensitivity to small changes in Mg²⁺ as demonstrated in cell-free extracts [31, 36, 37]. However, a common attitude seemed to be that intracellular Mg²⁺ is very well buffered and the cell goes to “some pains to hold [it] steady” because it influences so many reactions [38]. This attitude of course ignored the many reactions of the coordinate response that had to be balanced in order to obtain growth and proliferation, which makes Mg²⁺ the ideal second messenger for the response. It also ignored the papers showing there is a long term increase in Mg with growth factor treatment, e.g. [15], and failed to predict the increase in free Mg²⁺ demonstrated when specific fluorescent indicators became available [20, 21, 39].

Another criticism was that growth-stimulated cells do not exhibit a steep surge of Mg²⁺, as they do of Ca²⁺, so they are “not set up to use Mg²⁺ as a natural trigger” [40]. In this respect however, the relatively small increase in Mg²⁺ sustained over many hours parallels the increase in protein synthesis induced by growth factors, while the brief trigger-like increase of Ca²⁺ is incompatible with those effects. A further criticism of Mg²⁺ as regulator of the growth response was that Mg²⁺ is an essential rather than a regulatory element of the response [41]. This criticism is without foundation, however, because the level of free Mg²⁺ is set at a point that small increases or decreases are nowhere near the levels that threaten cellular metabolism, but they do affect the rate of coordinate response in a physiological manner. It is apparent that there was, and possibly still is, a misconception of the nature of the long term, finely graded nature of the coordinate response and particularly the rate of protein synthesis that makes Mg²⁺ the ideal candidate for the job.

It remained to be determined what specific biochemical process was activated by Mg²⁺ to accelerate the initiation pf protein synthesis. The precise mechanism, however, had to await the elucidation of the molecular pathway leading from the receptor binding of growth factors through intermediate steps to the increased frequency of translation. Understanding of such a pathway began to take shape in the late 1980s when the molecular era of growth regulation commenced. One of the best understood pathways for growth regulation is that instituted by the binding of insulin to its surface receptor and the activation of its tyrosine kinase function within the cell. This sets off the phosphatidyl inositol 3-kinase (PI-3K) pathway with its cascade of kinases that activates mTOR, a PI-3K related kinase. mTOR then phosphorylates two proteins that directly control the initiation of protein synthesis. All the kinases in the PI-3K pathway except mTOR have a very low requirement for ATP [42] and hence for free Mg²⁺. The requirement of mTOR for ATP is almost two orders of magnitude higher than that of the other kinases of the pathway. It was originally proposed that mTOR activity is dependent on the level of ATP in the cell, which was claimed to increase with cell stimulation by insulin [42]. However, ATP actually decreases slightly with cell stimulation [43]. Since the free Mg²⁺ level in the cell does increase [20, 21] and therefore increases MgATP^2-, which is the true substrate for kinases, it is assumed that the free Mg²⁺ level is the regulatory agency for protein synthesis. Thus the coordinate physiological response and the molecular aspects of PI-3K pathway are joined through the agency of Mg²⁺, consistent with the MMM model of cell proliferation control [2].

Acknowledgements

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