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NFATC2 transcription factor regulates cell cycle progression during lymphocyte activation: evidence of its involvement in the control of cyclin gene expression¹

MAURICIO S. CAETANO^*,2, ADRIANA VIEIRA-DE-ABREU^*,3, LEONARDO K. TEIXEIRA^*, MIRIAM B.F. WERNECK^*, MARCELLO A. BARCINSKI^*, and JOO P. B. VIOLA^*4

^* Division of Experimental Medicine, Brazilian National Cancer Institute, Rio de Janeiro, RJ, Brazil; and
Department of Parasitology, São Paulo University, São Paulo, SP, Brazil

⁴Correspondence: Divisão de Medicina Experimental, Instituto Nacional de Câncer (INCA), Praça Cruz Vermelha, 23 - 6^o andar, Rio de Janeiro, RJ, Brazil 20230-130. E-mail: jpviola{at}inca.gov.br

SPECIFIC AIMS

As previously demonstrated, NFATC2-/- mice consistently showed a marked increase in lymphocyte proliferation. In this work we evaluate the role of NFATC2 in regulating lymphocyte proliferation and its involvement in the control of cell cycle progression during lymphocyte activation.

PRINCIPAL FINDINGS

1. NFATC2-/- hyperproliferation is independent of antigen presenting cells (APC) and is not restricted to a specific lymphocyte subpopulation
Lymphocytes from NFATC2-/- mice proliferate more in an antigen dose-dependent manner than lymphocytes from NFATC2+/+ mice. NFATC2-/- lymphocytes also hyperproliferate compared with NFATC2+/+ lymphocytes after a polyclonal stimulation that bypasses antigen presentation, such as anti-CD3 antibody or PMA plus ionomycin. These results demonstrate that the hyperproliferative phenotype presented by NFATC2-/- cells is dependent on stimulation, but might be independent of antigen presentation and costimulation provided by APC. To better characterize the hyperproliferative phenotype presented by NFATC2-/- mice, we analyzed lymphocyte subsets that might be involved with this phenomenon. On in vitro stimulation with antigen, all lymphocyte subsets, including CD4⁺, CD8⁺, and B cells, were increased in NFATC2-/- cultures compared with NFATC2+/+ cultures. In the first 24 h the most significant difference was observed in CD4⁺ cells. These results suggest that the hyperproliferative phenotype presented by NFATC2-/- cells is not restricted to a specific lymphocyte subpopulation, however, CD4⁺ cells could play a major role in this phenomenon.

2. Hyperproliferation phenotype of NFATC2-/- lymphocyte is independent of IL-4 overexpression
It has been shown that CD4⁺ T cells from NFATC2-/- mice present preferential differentiation through a Th2 phenotype characterized by an overexpression of IL-4. Since IL-4 is a potent growth factor for Th2 and B cells, we evaluated the involvement of IL-4 in the hyperproliferation observed in NFATC2-/- lymphocytes. Lymphocytes from NFATC2-/- mice produce more IL-4 after in vitro antigen stimulation than NFATC2+/+ lymphocytes. However, proliferation assays demonstrated that NFATC2-/- cells still proliferate more than NFATC2+/+ cells even in the presence of neutralizing anti-IL-4 antibody. This demonstrates that the hyperproliferation observed in NFATC2-/- lymphocytes is IL-4 independent.

3. NFATC2-/- lymphocytes present an increased rate of apoptosis
On activation, T cells proliferate and die by apoptosis. This active form of apoptosis requires T cell receptor stimulation and involves death signals, including Fas/FasL engagement. Lymphoproliferative disorders have been linked to defects in T cell apoptosis in mice that possess nonfunctional Fas or FasL genes. Since NFAT transcription factors regulate expression of the FasL gene, one explanation for the hyperproliferative phenotype of NFATC2-/- mice would be a defect in apoptosis of NFATC2-/- lymphocytes. To address this question, cell death was analyzed. Surprisingly, NFATC2-/- cultures contained an increased number of dead cells after in vitro stimulation with antigen compared with NFATC2+/+ cultures. NFATC2-/- cultures also contained more annexin-V positive cells than NFATC2+/+ cultures on antigen stimulation. These results demonstrate that the lymphocyte hyperproliferation of NFATC2-/- mice is not related to diminished apoptosis and, conversely, that NFATC2-/- cells show an enhanced cell death rate as well as hyperproliferation.

4. NFATC2-/- lymphocytes present a deregulated cell cycle due to a shortening of time to cell division
On activation, lymphocytes initiate cell cycle progression and proliferate. Accumulating evidence indicates that active apoptosis is related to the cell cycle. Indeed, it has been shown that T cells are more susceptible to apoptosis in late G1 or S phase of the cell cycle. Based on these results, we asked whether hyperproliferation of NFATC2-/- lymphocytes might be due to cell cycle deregulation. Cell cycle analysis of lymph node cells stimulated in vitro with antigen demonstrated that NFATC2-/- cultures contained an increased cell number in S-G2/M phases vs. NFATC2+/+ cultures. These data suggested that the NFATC2-/- cell hyperproliferation might be related to an altered cell cycle in NFATC2-/- lymphocytes. One possibility would be that NFATC2-/- lymphocytes showed a shortening of time to cell division on activation. To investigate this hypothesis, we assessed cellular replication by analysis of NFATC2+/+ and NFATC2-/- lymphocytes labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). NFATC2-/- cultures contained cells in the first cell division after 42 h of in vitro stimulation with anti-CD3. NFATC2-/- cultures also contained more cells at 2, 3, and 4 divisions compared with NFATC2+/+ cultures after 65 h of stimulation. These results suggested that hyperproliferation of NFATC2-/- lymphocytes could be related to an altered cell cycle due to a shortening in time of cell division by these cells.

5. NFATC2-/- lymphocytes present an overexpression of cyclin genes on stimulation
The cell cycle is controlled by a family of protein kinases whose activities are regulated in response to cellular signals. Complexes of cyclin with cyclin-dependent kinase (CDK) play a central role in the control of cell cycle progression. Expression of specific cyclins dictate the formation of distinct cyclin/CDK complexes at different points of the cell cycle. Since cyclins are regulated at the gene expression level, they could represent important candidates for transcriptional control by the NFATC2 transcription factor. To investigate the role of NFATC2 in the expression of cyclin genes, we analyzed cyclin mRNA expression by RNase protection assay. As shown in Fig. 1 A, B, NFATC2-/- lymphocytes present an overexpression of cyclins A2, B1, E, and F compared with NFATC2+/+ lymphocytes after antigen stimulation. No differences in cyclin expression were observed in unstimulated cells from NFATC2+/+ and NFATC2-/- mice. Overexpression of these cyclins support the evidence that NFATC2-/- lymphocyte hyperproliferation is related to a deregulation in cell cycle control.

View larger version (33K): [in this window] [in a new window]   Figure 1. Analysis of cyclin expression of NFATC2+/+ and NFATC2-/- cells. Animals were sensitized and in vitro stimulated as described. Cells (2x107) were in vitro stimulated with OVA (0.5 mg/mL) for 24 h, collected, and total RNA was immediately extracted. A) Analysis of cyclins mRNA expression by RNase protection assay with a multiprobe assay. RNA loading was estimated by measuring the intensities of two housekeeping genes (L32 and GAPDH). Transcript levels were analyzed by autoradiography. B) Quantification of cyclins mRNA levels by densitometry. Values are expressed relative to L32 and GAPDH. The results are from a pool of 3 mice and are representative of 3 independent experiments.

DISCUSSION AND CONCLUSIONS
In this work we evaluated the role of the NFATC2 transcription factor in regulating cell cycle progression during lymphocyte activation. Our results demonstrated that NFATC2-/- lymphocytes display an enhanced proliferative response to antigen, suggesting that the NFATC2 transcription factor might exert an inhibitory effect on normal lymphocytes during antigen stimulation. Our results strongly support the idea that the hyperproliferative phenotype observed in NFATC2-/- lymphocytes is dependent on a cell-intrinsic mechanism and that the NFATC2 transcription factor is a key regulator of the responsiveness of lymphocytes to antigen. On antigen stimulation, NFATC2-/- mice present more cells in S-G2/M phases of the cell cycle than NFATC2+/+ mice. CFSE analysis demonstrated that NFATC2-/- lymphocytes present an increased rate of cellular replication. These data suggested that the NFATC2-/- cells present a deregulated cell cycle that could be due to a shortened cell cycle. NFATC2-/- lymphocytes present an overexpression of cyclins A2, B1, E, and F on stimulation vs. NFATC2+/+ lymphocytes. These data support the evidence that NFATC2-/- lymphocytes may present a shortening time of cell division that could be mediated by the overexpression of cyclins A2 and E.

There are conflicting results about the involvement of the cell cycle in Th differentiation. It has been suggested that IL-4 expression in Th2 cells is dependent on cell cycle progression. However, other data suggested that the cell cycle is important for the commitment of Th phenotype during the response to stimulation but not for IFN- or IL-4 expression. It has been shown recently that cell division plays a role in the frequency of IFN-- or IL-4-producing cells but is not essential for differentiation of Th lymphocytes. Although NFATC2-/- mice present a hyperproliferative response as well as an overexpression of Th2 cytokines, our results are not conclusive about the involvement of the cell cycle in Th differentiation. However, we can hypothesize that the expression of genes involved in cell cycle control and Th differentiation could be regulated in a coordinate way. The phenotype observed in NFATC2-/- lymphocytes supports this idea. Since NFATC2 is a preexisting transcription factor, this protein has an early effect on lymphocyte activation. Thus, we can speculate that NFAT transcription factors could simultaneously control expression of early inducible genes that are related to Th differentiation, cell death by apoptosis, and the cell division "clock" (Fig. 2 ). In conclusion, our data provide strong evidence that the NFATC2 transcription factor could play an important role in cell cycle control during lymphocyte activation by regulating cyclin expression. They also suggest that the NFAT transcription factor acts as a ubiquitous regulator of gene expression that might be reflected in lymphocyte activation.

View larger version (46K): [in this window] [in a new window]   Figure 2. Schematic diagram summarizing the involvement of NFAT transcription factors in regulating the expression of different genes during lymphocyte activation that are related to cell proliferation (cell cycle genes), Th cytokines (cell differentiation genes), and apoptosis (cell death genes). TCR, T cell receptor; BCR, B cell receptor; Ca2+, calcium; CaM, calmodulin; Cn, calcineurin; CsA, cyclosporin-A; NFAT, Nuclear Factor of Activated T cell; TFs, transcription factors; P, phosphorylation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0282fje; to cite this article, use FASEB J. (October 4, 2002) 10.1096/fj.02-0282fje

² Present address: Laboratory of Immunity Biology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil.

³ Present address: Laboratory of Immunopharmacology, Department of Physiology and Pharmacodynamics, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ, Brazil.

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