[Frontiers in Bioscience 3, d125-135, January 15, 1998]

	[Frontiers in Bioscience 3, d125-135, January 15, 1998] Reprints PubMed CAVEAT LECTOR
	DOES CALORIC RESTRICTION ALTER IL-2 TRANSCRIPTION? Mohammad A. Pahlavani Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System and Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78284 Received 1/5/98 Accepted 1/9/98 4. CALORIC RESTRICTION AND IL-2 TRANSCRIPTION To gain a better understanding of how caloric restriction affects IL-2 gene expression, our laboratory has focused its attention on transcriptional regulation of the IL-2 gene. Initially, our studies employed a nuclear run-off assay to ask directly if age altered the amount of nuclear transcription of the IL-2 gene. We showed that the induction of transcription by nuclei isolated from T cells was depressed with age; this decrese was proportional to the decline in IL-2 mRNA levels (37). More recently, we measured the nuclear transcription of IL-2 in 24-month-old caloric restricted rats and ad libitum fed rats and found that the induction of transcription by nuclei was higher (by approximately 40%) in T cells isolated from caloric restricted rats than in T cells isolated from ad libitum fed rats. How does caloric restriction specifically alter the transcription of genes? Caloric restriction does not appear to alter transcription through a general alteration in the transcriptional apparatus of a cell, since not all genes are affected in the same way by caloric restriction, e.g., the expression of some genes is increased, and the expression of others is decreased or not altered by caloric restriction (reviewed in 38). Thus, caloric restriction must alter transcription through a mechanism that affects only certain genes. It is currently known that transcription requires the recognition of numerous DNA sequences (cis-elements) by a diverse group of proteins, which are termed transcription factors. Transcription factors represent one of the largest and most diverse classes of DNA-binding proteins, and they regulate gene expression at the level of transcription. Over the past decade, it has become evident that these proteins play a critical role in development, differentiation and cellular proliferation (39-41). Transcription factors form a complex with RNA polymerase that initiates the synthesis of RNA. The assembly of the transcription complex requires that the DNA in the chromatin be accessible to the transcription factors, RNA polymerase, and the progression of the RNA polymerase along the DNA. Alteration in the expression and/or localization of transcription factors can result in changes in gene expression that would affect only one gene or a group of genes. Thus, changes in the activities or levels of transcription factors could be a mechanism whereby caloric restriction alters the transcription of genes in a specific manner. We have postulated that caloric restriction alters a transcription factor that plays a critical role in the regulation of the IL-2 gene. The transcription of the IL-2 gene is regulated by the binding of several transcription factors (NFAT, AP-1, AP-3, NF-kB, and OCT-1) to enhancer sequences within the 300-bp promoter region of the IL-2 gene (reviewed in 42,43). The transcription factors AP-1, AP-3, NF-kB, and OCT-1 are ubiquitous transcription factors; i.e., they are involved in regulation of a variety of genes in various tissues, whereas NFAT is an IL-2-specific transcription factor that is unique to T cells and binds to the NFAT purine-rich sequence in the IL-2 promoter (42,43). The NFAT transcription factor is a multipeptide complex consisting of a cytoplasmic component (NFAT-c) and constitutive and inducible nuclear component (NFAT-n) (44). The constitutive factor consists of members of a family of oncoproteins, i.e., Elf-1 (45). The inducible nuclear component consists of members of the Fos and Jun family of oncoproteins (46-49). Stimulation of T cells with an antigen/mitogen or phorbol ester induces the expression of the nuclear component of the NFAT complex, specifically Fos and Jun, through the protein kinase C (PKC) signaling pathway. In addition, an antigen, mitogen or calcium ionophore stimulates the translocation of NFAT-c from the cytoplasm into the nucleus through the inositol-1,4,5-triphosphate (IP3) signal transduction pathway. The cellular levels of calcium are elevated in the activated T cells, and it is believed that the increase in calcium levels activates the calcium-dependent phosphatase activity of calcineurin, which dephosphorylates NFAT-c (figure 2). The dephosphorylated form of NFAT-c then translocates into the nucleus and forms a complex with the nuclear components (Fos/Jun/Elf-1) of the NFAT complex. Binding of the NFAT complex (NFAT-c + NFAT-n) to the IL-2 promoter then stimulates the transcription of the IL-2 gene. Figure 2. Schematic illustration of NFAT activation by T cell receptor-mediated signal transduction pathways that lead to IL-2 transcription. Using nuclear extracts isolated from mitogen-stimulated T cells from rats fed either ad libitum or a caloric restricted diet, we measured the induction of DNA binding activity of the Tcell/IL-2-specific transcription factor NFAT and the ubiquitous transcription factor AP-1 by a gel shift assay. As the data in figure 3 show, the binding activity of NFAT was significantly higher in nuclear extracts from T cells isolated from rats fed a caloric restricted diet. In contrast to NFAT, the AP-1 binding activity in the nuclear extracts of T cells isolated from caloric restricted rats and ad libitum fed rats was not significantly different (33). In addition, the data in figure 3 also show that the increase in DNA binding activity of the transcription factor NFAT by caloric restriction was closely correlated to the increase in the transcription of the IL-2 gene (IL-2 activity and mRNA levels). Thus, it appears that caloric restriction alters the transcription of IL-2 through changes in the transcription factor NFAT. Figure 3. Influence of caloric restriction on the induction of IL-2 activity and mRNA levels and DNA binding activity of the transcription factors NFAT and AP-1 by Con A in T cells from F344 rats. Splenic T cells were isolated from 24-month-old rats fed ad libitum (AL) or 24-month-old rats fed a caloric restricted (CR) diet and were stimulated with Con A. IL-2 activity in the culture supernatants was measured by an IL-2-dependent cell line (CTLL-20) and the Con A induction of IL-2 mRNA levels was measured by Northern blot hybridization. The induction of the NFAT and AP-1 binding activity of the nuclear extracts were measured by the gel mobility shift assay. The IL-2 mRNA blots and the autoradiographs of the NFAT and AP-1 binding activities were quantified by Molecular Dynamic PhosphorImager, and the data are presented in the graph. Data were taken from Pahlavani et al. (33). The values () for the caloric restricted rats are significantly different from the values for the rats fed ad libitum* at p<0.05. Because Fos and Jun proteins are constituents of both the nuclear component of the NFAT protein complex (46-49) and the transcription factor AP-1 (42,43), we focused our attention on determining whether caloric restriction alters c-fos and/or c-jun expression. Figure 4 shows the effect of caloric restriction on the ability of T cells to express c-fos and c-jun after mitogen stimulation. The induction of c-fos expression (protein and mRNA levels) was significantly higher in T cells isolated from caloric restricted rats than from rats fed ad libitum (33). In contrast to c-fos, the c-jun expression was similar in caloric restricted and ad libitum fed rats. Thus, our study indicated that caloric restriction has a differential effect on c-fos and c-jun expression. Figure 4. Influence of caloric restriction on the induction of c-fos and c-jun expression by Con A in F344 rats. The splenic T cells were isolated from 24-month-old rats fed ad libitum (AL) or rats fed a caloric restricted (CR) diet and were stimulated with Con A. The induction of Fos and Jun protein levels were measured by Western blot analysis and c-fos and c-jun mRNA levels were measured by Northern blot hybridization. The blots were quantified by Molecular Dynamic PhosphorImager, and the data are presented in the graph. Data were taken from Pahlavani et al. (33). The values () for the caloric restricted rats are significantly different from the values for the rats fed ad libitum* at p<0.05.

[Frontiers in Bioscience 3, d125-135, January 15, 1998]
Reprints
PubMed
CAVEAT LECTOR

DOES CALORIC RESTRICTION ALTER IL-2 TRANSCRIPTION?

Mohammad A. Pahlavani

Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System and Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78284

Received 1/5/98 Accepted 1/9/98

4. CALORIC RESTRICTION AND IL-2 TRANSCRIPTION

To gain a better understanding of how caloric restriction affects IL-2 gene expression, our laboratory has focused its attention on transcriptional regulation of the IL-2 gene. Initially, our studies employed a nuclear run-off assay to ask directly if age altered the amount of nuclear transcription of the IL-2 gene. We showed that the induction of transcription by nuclei isolated from T cells was depressed with age; this decrese was proportional to the decline in IL-2 mRNA levels (37). More recently, we measured the nuclear transcription of IL-2 in 24-month-old caloric restricted rats and ad libitum fed rats and found that the induction of transcription by nuclei was higher (by approximately 40%) in T cells isolated from caloric restricted rats than in T cells isolated from ad libitum fed rats.

How does caloric restriction specifically alter the transcription of genes? Caloric restriction does not appear to alter transcription through a general alteration in the transcriptional apparatus of a cell, since not all genes are affected in the same way by caloric restriction, e.g., the expression of some genes is increased, and the expression of others is decreased or not altered by caloric restriction (reviewed in 38). Thus, caloric restriction must alter transcription through a mechanism that affects only certain genes. It is currently known that transcription requires the recognition of numerous DNA sequences (cis-elements) by a diverse group of proteins, which are termed transcription factors. Transcription factors represent one of the largest and most diverse classes of DNA-binding proteins, and they regulate gene expression at the level of transcription. Over the past decade, it has become evident that these proteins play a critical role in development, differentiation and cellular proliferation (39-41). Transcription factors form a complex with RNA polymerase that initiates the synthesis of RNA. The assembly of the transcription complex requires that the DNA in the chromatin be accessible to the transcription factors, RNA polymerase, and the progression of the RNA polymerase along the DNA. Alteration in the expression and/or localization of transcription factors can result in changes in gene expression that would affect only one gene or a group of genes. Thus, changes in the activities or levels of transcription factors could be a mechanism whereby caloric restriction alters the transcription of genes in a specific manner.

We have postulated that caloric restriction alters a transcription factor that plays a critical role in the regulation of the IL-2 gene. The transcription of the IL-2 gene is regulated by the binding of several transcription factors (NFAT, AP-1, AP-3, NF-kB, and OCT-1) to enhancer sequences within the 300-bp promoter region of the IL-2 gene (reviewed in 42,43). The transcription factors AP-1, AP-3, NF-kB, and OCT-1 are ubiquitous transcription factors; i.e., they are involved in regulation of a variety of genes in various tissues, whereas NFAT is an IL-2-specific transcription factor that is unique to T cells and binds to the NFAT purine-rich sequence in the IL-2 promoter (42,43). The NFAT transcription factor is a multipeptide complex consisting of a cytoplasmic component (NFAT-c) and constitutive and inducible nuclear component (NFAT-n) (44). The constitutive factor consists of members of a family of oncoproteins, i.e., Elf-1 (45). The inducible nuclear component consists of members of the Fos and Jun family of oncoproteins (46-49). Stimulation of T cells with an antigen/mitogen or phorbol ester induces the expression of the nuclear component of the NFAT complex, specifically Fos and Jun, through the protein kinase C (PKC) signaling pathway. In addition, an antigen, mitogen or calcium ionophore stimulates the translocation of NFAT-c from the cytoplasm into the nucleus through the inositol-1,4,5-triphosphate (IP3) signal transduction pathway. The cellular levels of calcium are elevated in the activated T cells, and it is believed that the increase in calcium levels activates the calcium-dependent phosphatase activity of calcineurin, which dephosphorylates NFAT-c (figure 2). The dephosphorylated form of NFAT-c then translocates into the nucleus and forms a complex with the nuclear components (Fos/Jun/Elf-1) of the NFAT complex. Binding of the NFAT complex (NFAT-c + NFAT-n) to the IL-2 promoter then stimulates the transcription of the IL-2 gene.

Figure 2. Schematic illustration of NFAT activation by T cell receptor-mediated signal transduction pathways that lead to IL-2 transcription.

Using nuclear extracts isolated from mitogen-stimulated T cells from rats fed either ad libitum or a caloric restricted diet, we measured the induction of DNA binding activity of the Tcell/IL-2-specific transcription factor NFAT and the ubiquitous transcription factor AP-1 by a gel shift assay. As the data in figure 3 show, the binding activity of NFAT was significantly higher in nuclear extracts from T cells isolated from rats fed a caloric restricted diet. In contrast to NFAT, the AP-1 binding activity in the nuclear extracts of T cells isolated from caloric restricted rats and ad libitum fed rats was not significantly different (33). In addition, the data in figure 3 also show that the increase in DNA binding activity of the transcription factor NFAT by caloric restriction was closely correlated to the increase in the transcription of the IL-2 gene (IL-2 activity and mRNA levels). Thus, it appears that caloric restriction alters the transcription of IL-2 through changes in the transcription factor NFAT.

Figure 3. Influence of caloric restriction on the induction of IL-2 activity and mRNA levels and DNA binding activity of the transcription factors NFAT and AP-1 by Con A in T cells from F344 rats. Splenic T cells were isolated from 24-month-old rats fed ad libitum (AL) or 24-month-old rats fed a caloric restricted (CR) diet and were stimulated with Con A. IL-2 activity in the culture supernatants was measured by an IL-2-dependent cell line (CTLL-20) and the Con A induction of IL-2 mRNA levels was measured by Northern blot hybridization. The induction of the NFAT and AP-1 binding activity of the nuclear extracts were measured by the gel mobility shift assay. The IL-2 mRNA blots and the autoradiographs of the NFAT and AP-1 binding activities were quantified by Molecular Dynamic PhosphorImager, and the data are presented in the graph. Data were taken from Pahlavani et al. (33). The values (*) for the caloric restricted rats are significantly different from the values for the rats fed ad libitum at p<0.05.

Because Fos and Jun proteins are constituents of both the nuclear component of the NFAT protein complex (46-49) and the transcription factor AP-1 (42,43), we focused our attention on determining whether caloric restriction alters c-fos and/or c-jun expression. Figure 4 shows the effect of caloric restriction on the ability of T cells to express c-fos and c-jun after mitogen stimulation. The induction of c-fos expression (protein and mRNA levels) was significantly higher in T cells isolated from caloric restricted rats than from rats fed ad libitum (33). In contrast to c-fos, the c-jun expression was similar in caloric restricted and ad libitum fed rats. Thus, our study indicated that caloric restriction has a differential effect on c-fos and c-jun expression.

Figure 4. Influence of caloric restriction on the induction of c-fos and c-jun expression by Con A in F344 rats. The splenic T cells were isolated from 24-month-old rats fed ad libitum (AL) or rats fed a caloric restricted (CR) diet and were stimulated with Con A. The induction of Fos and Jun protein levels were measured by Western blot analysis and c-fos and c-jun mRNA levels were measured by Northern blot hybridization. The blots were quantified by Molecular Dynamic PhosphorImager, and the data are presented in the graph. Data were taken from Pahlavani et al. (33). The values (*) for the caloric restricted rats are significantly different from the values for the rats fed ad libitum at p<0.05.