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(Circulation Research. 2000;87:638.)
© 2000 American Heart Association, Inc.

Editorial

GATA-6: The Proliferation Stops Here

Cell Proliferation in Glomerular Mesangial and Vascular Smooth Muscle Cells

Edward E. Morrisey

From the Department of Medicine, University of Pennsylvania, Philadelphia, Pa.

Correspondence to Edward E. Morrisey, PhD, Department of Medicine, University of Pennsylvania, 953 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104. E-mail emorrise{at}mail.med.upenn.edu

Key Words: cyclin-dependant kinase inhibitors • transcription factors • cell cycle • differentiation

	Introduction

Top Introduction References

 
See related article, pages 699–704

GATA-6 belongs to a family of zinc finger transcription factors that have been implicated in various developmental processes, including cell differentiation and proliferation. In this issue of Circulation Research, Nagata et al¹ report that overexpression of GATA-6 in glomerular mesangial cells (GMCs) results in decreased cell proliferation and posttranscriptional upregulation of p21^cip1, a cyclin-dependent kinase (cdk) inhibitor. This finding is especially interesting in light of recent reports showing that GATA-6 regulates cell proliferation via p21^cip1 in vascular smooth muscle cells (VSMCs).² Thus, the present study details a new region of expression for GATA-6 (glomerular mesangial cells) coupled with an important emerging function (regulation of cell proliferation).

All GATA factors activate transcription by binding to the consensus DNA sequence WGATAR (where W is T or A and R is G or A).^{3 4} On the basis of both their amino acid sequence homologies and respective patterns of expression, the 6 vertebrate GATA factors have been classified into 2 subfamilies. Members of the GATA-1/-2/-3 subfamily are known to be important regulators of hematopoietic cell growth and differentiation.⁵ In addition, GATA-1 has been shown to regulate erythroid cell proliferation, whereas GATA-2 seems to regulate multipotential hematopoietic progenitor cell proliferation.^{6 7} GATA-6 was originally characterized as a member of the GATA-4/-5/-6 subfamily of GATA factors, which are expressed in the developing heart and are able to transactivate cardiac-specific promoters, such as cardiac troponin C, atrial natriuretic factor, and -myosin heavy chain, in vitro.⁸ Recent reports of mice containing null mutations for GATA-4 and -6 have shown that these factors are essential for formation of the midline heart tube from precardiac mesoderm and differentiation of the visceral endoderm in the mouse, respectively.^{9 10 11} Although a great deal has been learned about the roles that GATA factors play during development from gene knockout experiments, much is still not understood about the molecular targets and mechanisms by which these factors influence tissue development, cell differentiation, and proliferation.

Excessive proliferation of GMCs is associated with glomerulonephritis, which, in extreme cases, can lead to renal hypertension. Therefore, the ability to control GMC proliferation would be of great potential therapeutic value in the treatment of this disease. Although in recent years there have been many reports characterizing the mechanisms of GMC proliferation, very little is known about the tissue-specific regulation of growth and proliferation in these cells. However, from these studies, two different and widely expressed transcription factors have emerged as important regulators of GMC proliferation: early response growth factor-1 (Egr-1) and E2F1. Egr-1 is a zinc finger transcription factor that is induced under a variety of mitogenic signals and during differentiation of certain cell lineages.¹² Treatment of GMCs with antisense oligonucleotide directed against Egr-1 inhibits cell proliferation by up to 46%.¹³ In addition, stimulation of nitric oxide production in GMCs inhibited their growth by disrupting the ability of Egr-1 to bind to DNA.¹⁴ Together, these data suggest that Egr-1 probably plays a direct and positive role in regulating GMC growth and proliferation. E2F1 has also been implicated in regulating cell proliferation in GMCs. E2F proteins bind to the retinoblastoma tumor-suppressor family, and, in this complex, they remain inactive. Phosphorylation of the retinoblastoma protein releases and activates E2F proteins that then induce target genes essential for the G1- to S-phase transition of the cell cycle.¹⁵ Members of the E2F family regulate the expression of a variety of cell cycle genes, including cdk2, c-myb, c-myc, and proliferating cell nuclear antigen. In addition, E2F1 binding sites are found in the promoters of other cell cycle–dependent genes, such as cyclin D1 and E, additionally supporting its role as a regulator of cell cycle progression.¹⁵ The level of E2F1 protein is undetectable in quiescent GMCs but increases dramatically after serum stimulation, reaching a maximum at 24 hours.¹⁶ In addition, overexpression of E2F1 in rat GMCs results in an increase of thymidine uptake of up to 250%, with a subsequent increase in cell proliferation.¹⁷ These data indicate that the E2F family, in particular E2F1, plays an important role in GMC cell cycle regulation by promoting the progression of cells through the G1/S transition. Although the results described here with Egr-1 and E2F1 are intriguing, one must keep in mind that both of these transcription factors are expressed ubiquitously and have been demonstrated to affect proliferation in many other cell types. Consequently, any therapy based on inhibiting these proteins would most likely have severe deleterious effects on other cells and tissues. Therefore, transcription factors (or other cell cycle regulatory proteins) that are more tissue-restricted may provide better targets for regulating GMC growth and proliferation.

Because GMCs share many similarities with VSMCs, as Nagata et al¹ correctly point out in their study, a discussion of the mechanisms of cell proliferation in VSMCs is beneficial to understanding GMC proliferation. There has been great interest in characterizing the mechanisms of VSMC proliferation, because this is a major cause of arterial occlusion after vessel injury. From several studies over the past few years, the transcription factors Gax and GATA-6 have emerged as candidate regulators of VSMC proliferation. Gax was originally identified as a homeodomain protein expressed at high levels in nonproliferating VSMCs whose expression decreased dramatically on entry of these cells into the cell cycle.¹⁸ Later reports showed that forced expression of Gax inhibited cell proliferation in both VSMCs and fibroblasts, indicating that Gax may have a general antiproliferative effect in cells other than VSMCs.¹⁹ Whether Gax is expressed in GMCs is not known, but this should be investigated due to its role in VSMC proliferation. The expression of GATA-6 also decreases dramatically on entry of VSMCs into the cell cycle, and, like Gax, forced expression of GATA-6 in VSMCs results in decreased cell proliferation.² Interestingly, the inhibition of cell proliferation demonstrated by both Gax and GATA-6 seems to be mediated by p21^cip1, because p21^cip1-deficient fibroblasts were not affected by overexpression of either transcription factor. In addition, overexpression of Gax or GATA-6 induced cell cycle arrest in p53-deficient fibroblasts, indicating that they directly modulate the expression of p21^cip1 independent of p53, which itself can activate p21^cip1.

These reports suggest that Gax and GATA-6 may be involved in the modulation of VSMC differentiation and, by association, GMC differentiation. VSMCs are able to modulate their phenotype by dedifferentiating from the contractile nonproliferative state and switching to a proliferative noncontractile state. Since the expression levels of Gax and GATA-6 decrease rapidly on entry of VSMCs into the cell cycle, either or both may be responsible for maintaining VSMCs in their normal contractile and quiescent phenotype. Obviously, this is an important area of investigation, because modulating the activity of these proteins could lead to a better understanding of VSMC proliferation (and, by association, GMC proliferation) and provide better targets for drug therapy. This is clearly a point that Nagata et al¹ and other investigators will want to explore. However, caution is needed before drawing too many conclusions from these studies, because both Gax and GATA-6 have similar effects on fibroblasts and VSMCs, suggesting that their effects are more global and less tissue-restricted than they may seem at first glance. Despite these uncertainties, Gax and GATA-6 presently seem to be the best candidates for tissue-specific transcription factors that may regulate VSMC proliferation, primarily because of their restricted expression patterns. In this regard, it will be interesting to determine whether Gax or GATA-6 controls cell proliferation in tissues other than VSMCs where they are expressed such as cardiac myocytes, which are terminally differentiated and, thus, unable to reenter the cell cycle.

From the background data discussed above, it can be postulated that GATA-6 plays an important role in the regulation of VSMC proliferation. In the present report by Nagata et al,¹ the authors have logically decided to examine whether GATA-6 also regulates cell proliferation in GMCs. As was observed in VSMCs, GATA-6 expression was detected in quiescent GMCs but decreased dramatically on serum stimulation. In addition, forced expression of GATA-6 inhibited cell proliferation in GMCs in a p21^cip1-dependant manner. What is unique about this study¹ and earlier work² by Nagata and colleagues on VSMCs is that the regulation of p21^cip1 by GATA-6 is at the posttranscriptional level. Nagata et al¹ show that GATA-6, by an uncharacterized mechanism, seems to stabilize the p21^cip1 protein. The stability of p21^cip1 protein levels was recently shown to be regulated by the ubiquitin-proteasome pathway.²⁰ These studies also showed that interactions with Cdks decrease p21^cip1 stability, whereas interaction with proliferating cell nuclear antigen increases the stability of p21^cip1. Interestingly, the CCAAT/enhancer binding protein also binds to p21^cip1.²¹ This interaction results in an inhibition of the ubiquitin-mediated proteolysis of p21^cip1. Thus, interactions with a variety of nuclear proteins regulate p21^cip1 protein stability. Whether GATA-6 physically interacts with p21^cip1 and inhibits its ubiquitin-mediated proteolysis is unknown but should be examined in future investigations. Regardless of the mechanism, the ability of GATA-6 to stabilize p21^cip1 protein levels should be explored in greater detail, since it may highlight a new role for GATA-6 outside of its role in direct transcriptional regulation.

In addition to the stabilization of p21^cip1, GATA-6 also inhibited the expression of cyclin A, but not cyclin D1 or E. This is an observation that was not reported by Nagata and colleagues in their previous work² on VSMCs, and, as such, it is not known whether this result is unique to GMCs. However, because GATA-6 normally acts as a transcriptional activator, it will be of interest to determine the mechanism by which it represses cyclin A expression. In this regard, it is noteworthy that GATA factors interact with a family of transcriptional repressors called Friends of GATA (FOG).^{22 23} Future studies should determine whether the known FOG family members, FOG-1 and -2, are expressed in GMCs and whether they participate in the regulation of cyclin A.

In conclusion, the inhibition of cell proliferation in GMCs by overexpression of GATA-6 is a new chapter in an ever-growing volume on the diverse roles that GATA factors play during development. Although it appears from these studies that one of the major functions of GATA-6 in GMCs and VSMCs is regulating cell proliferation, the mechanism by which GATA-6 stabilizes the p21^cip1 protein still needs to be determined. In addition, it will be of great interest to determine additional downstream transcriptional targets of GATA-6 in GMCs (and VSMCs), since this may provide additional clues as to how GATA-6 may negatively regulate cell proliferation. However, investigators should keep in mind that much of the data discussed above are derived from in vitro studies and, therefore, will need to be confirmed by additional investigations in vivo. In the end, tissue restricted knockouts in mice will need to be generated and analyzed to fully determine the role that GATA-6 plays during the growth and development of GMCs and VSMCs.

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

	References

Top Introduction References

 

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