[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 5. SIGNAL TRANSDUCTION AND IL-2 EXPRESSION The upstream signaling pathways involve a cascade of phosphorylation and dephosphorylation events which lead to augmentation of c-fos and c-jun transcription and ultimately IL-2 expression. Therefore, in order to gain insight into the mechanisms responsible for the changes in NFAT and IL-2 with age and caloric restriction, we have been interested in studying the activation of the upstream signaling molecules. Among various signal transduction molecules, the mitogen-activated protein kinase (MAPK), also known as extracellular regulated kinase (ERK), and the c-jun amino terminal kinase (JNK) have been shown to play an integral role in transduction of receptor-mediated signals in T cells (50-52). The activation of MAPK/JNK has been shown to be an important regulatory signal through which a wide variety of extracellular signals are transduced into the intracellular events. In Jurkat T cells, at least two isoforms of MAPKs, ERK1 (p44^mapk) and ERK2 (p42^mapk) are present. These isoforms are transiently activated by various stimuli, including Con A, phytohemagglutinin (PHA), phorbol myristate acetate (PMA), and anti-CD3 mitogenic antibody. It has been shown that over-expression of ERK1 enhances the induction of IL-2, probably through increasing the activity of the transcription factors NFAT and AP-1 (53). Thus, these studies suggest that stimulation of MAPK plays an important role in T cell activation and IL-2 expression. The current model for MAPK signaling events that leads to regulation of c-fos and c-jun transcription is shown in figure 5. In T cells, TCR (T cell receptor) signaling is mediated through protein tyrosine kinases (PTKs) activity although no tyrosine kinase domain has been identified within the TCR-CD3 structure. The TCR interacts with at least three PTKs (Lck, Fyn, and ZAP-70) through the tyrosine-based activation motif (TAM) contained in the z and other CD3 chains (52,54,55). Lck is usually not found physically associated with TCR, but it binds to the co-receptors CD4 and CD8. Lck does, however, interact with the TCR because CD4 and CD8 co-localize with this receptor during antigenic/mitogenic recognition (56). This clustering allows Lck to phosphorylate the TAM on the TCR z chain, leading to the recruitment of ZAP-70 (57). In contrast to ZAP-70, Fyn appears to bind directly to the TCR z chain without requiring prior receptor ligation (58). The tyrosine kinase activity associated with the TCR-associated molecules is coupled to the activation of downstream signalling molecules (52, 54-58). The tyrosine kinase phospholipase-Cg (PLC-g) stimulates its activity, causing the generation of second messengers that stimulate protein kinase C (PKC) activation and trigger an elevation of intracellular calcium. These biochemical events lead to the stimulation of GTP-bound Ras. Ras is activated both as a result of PKC mediated inhibition of Ras-GTPase-activating proteins, and by an additional stimulatory signal that is independent of PKC, and likely involves coupling of TCR-associated protein tyrosine kinase activity to Grb2 and the Ras Grb-exchange SOS molecule (50,51). In the GTP-bound state, Ras stimulates the MAPK pathway, leading to activation of ERKs and JNKs. At the plasma membrane, active GTP-bound Ras directly binds and promotes the activation of the protein kinase Raf-1. Active Raf-1 phosphorylates and activates the MAPKs/ERKs through the activation of MAPK kinase (MEK). Active ERKs phosphorylate and regulate the activity of numerous additional proteins in both the cytosol and nucleus (50-51). Thus, one function of the Ras/Raf-1/MEK/ERK signal transduction pathway is to transmit the stimulatory signal received at the plasma membrane into the nucleus. Figure 5. The current model for the intracellular signaling event involving the activation of the MAPK signal transduction pathway acting on c-fos and c-jun transcription and IL-2 expression. Given the potential important role of the upstream signaling molecules, i.e., MAPK in regulation of c-fos and c-jun transcription, and because Fos and Jun proteins constitute the nuclear component of the NFAT protein complex and the transcription factor AP-1, our laboratory has begun to study how caloric restriction affects the upstream signaling molecules. To determine whether aging or caloric restriction affects MAPK and/or JNK activation, purified T cells were isolated from young and old rats fed ad libitum and old rats fed a caloric restricted diet, and the kinase activity of the immunoprecipitated p44 and p42 MAPK, and p46 JNK were measured by the phosphorylation of myelin basic protein and recombinant GST-c-jun peptide substrate, respectively. As shown in figure 6, we found that mitogen induction of MAPK activity decreased with age, and caloric restriction reduced the age-related decrease in MAPK activity. For example, the MAPK activity was 65% higher for T cells isolated from 24-month-old caloric restricted rats compared to T cells isolated from age-matched control rats. On the other hand, the data in figure 6 show that JNK activity did not change significantly with age or with caloric restriction. The changes in MAPK/JNK activities were not associated with changes in their corresponding protein levels as measured using Western blot analysis. Thus, caloric restriction appears to increase the MAPK activity, and this increase was correlated with an increase in c-fos expression. Figure 6. Effect of age and caloric restriction on the induction of mitogen-activated protein kinase (MAPK) and c-jun amino terminal kinase (JNK) activities by Con A in T cells from F344 rats. The splenic T cells from young (6 months) and old (24 months) ad libitum fed rats and 24-month-old caloric restricted (CR) rats were stimulated with Con A for 15 min. Protein was isolated and kinase activity in the immunoprecipitated p44 and p42 MAPK or p46 JNK was measured by their ability to phosphorylate a myelin basic protein or a recombinant GST-c-jun exogenous substrate in the presence of ³²P-ATP. The ³²P-ATP incorporation into the myelin basic protein substrate was measured by scintillation counting. The ³²P-ATP incorporation into the GST-c-jun substrate was analyzed by SDS-PAGE and the autoradiographs were quantified and the data are presented in the graph. The kinase activity is expressed as the percent of activity in Con A-stimulated cells over the unstimulated cells. Each value represents mean ± SD of 3 separate experiments. The values () for the control young rats are significantly different from the values for the old rats fed ad libitum* at p<0.001. The values (*) for caloric restricted old rats were significantly different from the value for the old rats fed ad libitum* at p<0.05.

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

5. SIGNAL TRANSDUCTION AND IL-2 EXPRESSION

The upstream signaling pathways involve a cascade of phosphorylation and dephosphorylation events which lead to augmentation of c-fos and c-jun transcription and ultimately IL-2 expression. Therefore, in order to gain insight into the mechanisms responsible for the changes in NFAT and IL-2 with age and caloric restriction, we have been interested in studying the activation of the upstream signaling molecules. Among various signal transduction molecules, the mitogen-activated protein kinase (MAPK), also known as extracellular regulated kinase (ERK), and the c-jun amino terminal kinase (JNK) have been shown to play an integral role in transduction of receptor-mediated signals in T cells (50-52). The activation of MAPK/JNK has been shown to be an important regulatory signal through which a wide variety of extracellular signals are transduced into the intracellular events. In Jurkat T cells, at least two isoforms of MAPKs, ERK1 (p44^mapk) and ERK2 (p42^mapk) are present. These isoforms are transiently activated by various stimuli, including Con A, phytohemagglutinin (PHA), phorbol myristate acetate (PMA), and anti-CD3 mitogenic antibody. It has been shown that over-expression of ERK1 enhances the induction of IL-2, probably through increasing the activity of the transcription factors NFAT and AP-1 (53). Thus, these studies suggest that stimulation of MAPK plays an important role in T cell activation and IL-2 expression.

The current model for MAPK signaling events that leads to regulation of c-fos and c-jun transcription is shown in figure 5. In T cells, TCR (T cell receptor) signaling is mediated through protein tyrosine kinases (PTKs) activity although no tyrosine kinase domain has been identified within the TCR-CD3 structure. The TCR interacts with at least three PTKs (Lck, Fyn, and ZAP-70) through the tyrosine-based activation motif (TAM) contained in the z and other CD3 chains (52,54,55). Lck is usually not found physically associated with TCR, but it binds to the co-receptors CD4 and CD8. Lck does, however, interact with the TCR because CD4 and CD8 co-localize with this receptor during antigenic/mitogenic recognition (56). This clustering allows Lck to phosphorylate the TAM on the TCR z chain, leading to the recruitment of ZAP-70 (57). In contrast to ZAP-70, Fyn appears to bind directly to the TCR z chain without requiring prior receptor ligation (58). The tyrosine kinase activity associated with the TCR-associated molecules is coupled to the activation of downstream signalling molecules (52, 54-58). The tyrosine kinase phospholipase-Cg (PLC-g) stimulates its activity, causing the generation of second messengers that stimulate protein kinase C (PKC) activation and trigger an elevation of intracellular calcium. These biochemical events lead to the stimulation of GTP-bound Ras. Ras is activated both as a result of PKC mediated inhibition of Ras-GTPase-activating proteins, and by an additional stimulatory signal that is independent of PKC, and likely involves coupling of TCR-associated protein tyrosine kinase activity to Grb2 and the Ras Grb-exchange SOS molecule (50,51). In the GTP-bound state, Ras stimulates the MAPK pathway, leading to activation of ERKs and JNKs. At the plasma membrane, active GTP-bound Ras directly binds and promotes the activation of the protein kinase Raf-1. Active Raf-1 phosphorylates and activates the MAPKs/ERKs through the activation of MAPK kinase (MEK). Active ERKs phosphorylate and regulate the activity of numerous additional proteins in both the cytosol and nucleus (50-51). Thus, one function of the Ras/Raf-1/MEK/ERK signal transduction pathway is to transmit the stimulatory signal received at the plasma membrane into the nucleus.

Figure 5. The current model for the intracellular signaling event involving the activation of the MAPK signal transduction pathway acting on c-fos and c-jun transcription and IL-2 expression.

Given the potential important role of the upstream signaling molecules, i.e., MAPK in regulation of c-fos and c-jun transcription, and because Fos and Jun proteins constitute the nuclear component of the NFAT protein complex and the transcription factor AP-1, our laboratory has begun to study how caloric restriction affects the upstream signaling molecules. To determine whether aging or caloric restriction affects MAPK and/or JNK activation, purified T cells were isolated from young and old rats fed ad libitum and old rats fed a caloric restricted diet, and the kinase activity of the immunoprecipitated p44 and p42 MAPK, and p46 JNK were measured by the phosphorylation of myelin basic protein and recombinant GST-c-jun peptide substrate, respectively. As shown in figure 6, we found that mitogen induction of MAPK activity decreased with age, and caloric restriction reduced the age-related decrease in MAPK activity. For example, the MAPK activity was 65% higher for T cells isolated from 24-month-old caloric restricted rats compared to T cells isolated from age-matched control rats. On the other hand, the data in figure 6 show that JNK activity did not change significantly with age or with caloric restriction. The changes in MAPK/JNK activities were not associated with changes in their corresponding protein levels as measured using Western blot analysis. Thus, caloric restriction appears to increase the MAPK activity, and this increase was correlated with an increase in c-fos expression.

Figure 6. Effect of age and caloric restriction on the induction of mitogen-activated protein kinase (MAPK) and c-jun amino terminal kinase (JNK) activities by Con A in T cells from F344 rats. The splenic T cells from young (6 months) and old (24 months) ad libitum fed rats and 24-month-old caloric restricted (CR) rats were stimulated with Con A for 15 min. Protein was isolated and kinase activity in the immunoprecipitated p44 and p42 MAPK or p46 JNK was measured by their ability to phosphorylate a myelin basic protein or a recombinant GST-c-jun exogenous substrate in the presence of ³²P-ATP. The ³²P-ATP incorporation into the myelin basic protein substrate was measured by scintillation counting. The ³²P-ATP incorporation into the GST-c-jun substrate was analyzed by SDS-PAGE and the autoradiographs were quantified and the data are presented in the graph. The kinase activity is expressed as the percent of activity in Con A-stimulated cells over the unstimulated cells. Each value represents mean ± SD of 3 separate experiments. The values (*) for the control young rats are significantly different from the values for the old rats fed ad libitum at p<0.001. The values (**) for caloric restricted old rats were significantly different from the value for the old rats fed ad libitum at p<0.05.