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Endocrinology Vol. 141, No. 11 4202-4208
Copyright © 2000 by The Endocrine Society

ARTICLES

p38MAPK Inhibition Enhances Basal and Norepinephrine-Stimulated p42/44MAPK Phosphorylation in Rat Pinealocytes1

M. Mackova, J. R. Man, C. L. Chik and A. K. Ho

Department of Physiology (M.M., J.R.M., A.K.H.) and Department of Medicine (C.L.C.), Faculty of Medicine, University of Alberta, 7-26 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada

Address all correspondence and requests for reprints to: Dr. A. K. Ho, Department of Physiology, 7–26 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada. E-mail: anho{at}ualberta.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interaction between p38MAPK and p42/44MAPK in rat pinealocytes was examined by determining the effects of p38MAPK inhibitors on the phosphorylation of p42/44MAPK using Western blot analysis. Treatment with SB202190, a specific inhibitor of p38MAPK, increased p42/44MAPK phosphorylation in a concentration-dependent manner. SB202190 also enhanced the magnitude and the duration of norepinephrine-activated p42/44MAPK phosphorylation. The effect of SB202190 on p42/44MAPK phosphorylation was abolished by PD98059 or UO126, inhibitors of MEK, suggesting that SB202190 is acting upstream of MEK in activating p42/44MAPK. The SB202190-induced phosphorylation of p42/44MAPK was not blocked by inhibitors of cGMP-dependent kinase (KT5823), protein kinase C (calphostin C) or Ca2+/calmodulin dependent kinase (KN93) suggesting that these pathways may not be involved in the effect of SB202190. SB202190 further increased p42/44MAPK phosphorylation that was stimulated by 8-bromo-cGMP, 4ß phorbol 12-myristate 13-acetate, or ionomycin. In contrast, inhibition of p42/44MAPK phosphorylation by dibutyryl-cAMP persisted when p42/44MAPK phosphorylation was increased by SB202190. Furthermore, inhibition of p42/44MAPK phosphorylation had no effect on p38MAPK activation. These results suggest that inhibition of p38MAPK causes activation of p42/44MAPK and acts synergistically with norepinephrine in the regulation of p42/44MAPK activation in rat pinealocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MITOGEN-ACTIVATED protein kinases (MAPKs), which play an important role in mediating cellular responses to various extracellular stimuli (1, 2) can be divided into three subgroups: 1) extracellularly responsive kinases (p42/44MAPK or extracellular signal-regulated kinase1/2) (3, 4, 5); 2) c-Jun N-terminal kinases (p46/54JNK or stress-activated kinases) (6, 7), and 3) p38MAPK (8, 9, 10, 11). Whereas p42/44MAPK is typically stimulated by growth factors and mitogenic stimuli, p38MAPK and p46/54JNK are mainly activated by cellular stresses (11, 12). Although extracellular stimuli may be different, the mechanisms of activating different MAPK subfamilies are similar in that parallel phosphorylation cascades are involved which result in the activation of MAPKs through phosphorylation of threonine and tyrosine residues of the enzymes (13, 14). In the case of p42/44MAPK, the dual specific kinases responsible for their phosphorylation and activation are the MAPK kinase (MEK1/2) (15), whereas p38MAPK is activated by MAPK kinase 3 (MKK3) and MKK6, and p46/54JNK by MKK4 and MKK7 (16, 17).

Among the different MAPKs, p42/44MAPK is the most studied. Besides signaling pathways activated by growth factors, pathways involving cyclic nucleotides (18, 19), protein kinase C (PKC) (20, 21) and intracellular Ca2+ (22) have all been shown to modulate p42/44MAPK activation. More recently, fine tuning of cellular responses to MAPKs through interaction of p42/44MAPK and p38MAPK has also been reported (23, 24, 25, 26).

In rat pinealocytes, we have demonstrated the presence of high level of Raf-1, MEK1 and p42/44MAPK (27) and activation of p42/44MAPK by norepinephrine (NE) (27), the endogenous neurotransmitter that regulates pineal functions (28). The main signaling mechanism that mediates the effect of NE on p42/44MAPK phosphorylation is the cGMP/protein kinase G (PKG) pathway (27). The high level of expression of p38MAPK in rat pinealocytes (our unpublished observation) and known interaction between MAPK subfamilies in other cells (23, 24, 25, 26) suggest that potential interaction between p38MAPK and p42/44MAPK may modulate MAPK activation in the pineal gland. In the present study, we investigated the interaction between p38MAPK and p42/44MAPK by determining the effect of p38MAPK inhibition on basal and stimulated p42/44MAPK phosphorylation. Two specific inhibitors of p38MAPK, SB203580 and SB202190, were used and SB202474 was included as a negative control (29, 30).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
8-Bromo-cAMP, dibutyryl-cAMP, isoproterenol, NE, 4ß phorbol 12-myristate 13-acetate (PMA), polyclonal antibodies against p42/44MAPK and p38MAPK and monoclonal antibodies against phosphorylated p42/44MAPK (p-p42/44MAPK)and p38MAPK (p-p38MAPK) were obtained from Sigma (St. Louis, MO). SB202190, SB203580, SB202474, H89, H7, KT5823, KN93, calphostin C, UO124, UO126, PD98059, calyculin A, dephostatin and ionomycin were obtained from Calbiochem Corp. (San Diego, CA). All other chemicals were of the purest grades available commercially. Antibodies for the RIAs of cAMP and cGMP were gifts from Dr. A. Baukal (NICHHD, NIH, Bethesda, MD).

Preparation of pinealocytes
Sprague Dawley rats (male; weighing 150 g) were obtained from the University of Alberta Animal Unit. Pinealocytes were prepared from freshly dissected rat pineal glands by trypsinization as described previously (31, 32). The cells were suspended in DMEM containing 10% FCS and maintained at 37 C for 24 h in a gas mixture of 95% air and 5% CO2 before experiments. Tissue samples were freshly collected, rinsed in ice-cold PBS, and kept frozen in dry ice until homogenized in a buffer solution (20 mM Tris-HCl, pH 7.5 containing 2 mM EDTA, 5 mM EGTA, 1% Triton-X, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM sodium orthovanadate, and 2 mM phenylmethylsulphonyl fluoride). After centrifuged at 15,000 x g for 15 min at 4 C, the supernatant was assayed for protein and its concentration adjusted. After mixing with 2x sample buffer and boiled for 5 min, the samples were stored frozen until electrophoresis.

Drug treatment of pinealocytes
For the determination of MAPK activation, aliquots of pinealocytes (5 x 104 cells/0.5 ml for p42/44MAPK and 1 x 105 cells/0.5 ml for p38MAPK) were treated with drugs that had been prepared in concentrated solutions in water or dimethylsulfoxide for 15 min (unless otherwise indicated). Treated cells were collected by centrifugation (2 min, 12,000 x g). Samples for Western blot analysis were solubilized in 1x sample buffer (20 mM Tris-HCl, pH 6.8 containing 2 mM EDTA, 5 mM EGTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM sodium orthovanadate, and 2 mM phenylmethylsulfonyl fluoride; 5% 2-mercaptoethanol; 10% glycerol, 2% SDS and 0.002% bromphenol blue) by boiling for 5 min and stored until electrophoresis.

Western blot
SDS-PAGE was performed according to the procedure of Laemmli (33) using 10% acrylamide (Mini-Protein II gel system, Bio-Rad Laboratories, Inc., Hercules, CA). Following electrophoresis, gels were equilibrated for 20 min in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol). Proteins were transferred onto polyvinylidene difluoride membranes (1 h, 100 V) which were then incubated with a blocking solution [5% dried skim milk in 100 mM Tris (pH 7.5) with 140 mM NaCl and 0.01% Tween 20] for a minimum of 1.5 h. The blots were then incubated overnight at 4 C with diluted specific antisera as indicated. After washing twice with the blocking solution, the blots were incubated with diluted horseradish peroxidase-conjugated second antibodies (Bio-Rad Laboratories, Inc.) for 1 h at room temperature. They were then washed extensively and developed using enhanced chemiluminescence (Amersham Pharmacia Biotech).

cAMP and cGMP assays
cAMP and cGMP measurements were made on samples of cells (1.5 x 104 cells/0.4 ml) treated with different drugs for 15 min; the RIA method of detection has been described in detail (32, 34).

Statistical analysis
For the Western blots, a typical blot from at least three similar experiments was presented. Selected results were quantified using densitometric measurements and analyzed by Sigmagel (Jandel Scientific Software, San Rafael, CA), normalized to the control level and presented as the mean ± SEM from three separate experiments. Statistical comparisons were analyzed by paired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
p38MAPK expression in rat pineal gland
The expression of p38MAPK in the rat pineal gland was compared with those in other tissues including the cortex, hypothalamus, cerebellum, anterior pituitary, and liver. Western blot analysis showed that the level of p38MAPK expression in the rat pineal gland was comparable to hypothalamus, cortex, and cerebellum but less than that in the anterior pituitary gland or liver (Fig. 1Go).



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  		Figure 1. Tissue distribution of p38MAPK. Tissue protein (25 µg per lane) was subjected to 10% SDS-PAGE. p38MAPK was identified by Western blotting using polyclonal antibodies as described in Materials and Methods. COR, Cortex; HYP, hypothalamus; CER, cerebellum; PIN, pineal; PIT, pituitary; LIV, liver. The blot shown is representative of three separate experiments with similar results.

 
Effects of p38MAPK inhibitors on the activation of p42/44MAPK
Treatment of pinealocytes with SB202190, a specific p38MAPK inhibitor (29), for 15 min caused a concentration-dependent increase in p-p42/44MAPK with a significant increase observed at 1 µM of SB202190 (Fig. 2AGo). In contrast, the negative control SB202474 (10 µM) had no effect on p-p42/44MAPK. The maximal increase in p-p42/44MAPK by SB202190 (10 µM) treatment was observed at 15 min, and the increase was sustained for at least 60 min (Fig. 2BGo). Similar results were obtained with SB203580, another specific inhibitor of p38MAPK (30) (data not shown). To determine the involvement of MEK1/2 in SB202190-induced increase in p-p42/44MAPK, two MEK inhibitors, PD98059 and UO126, were used (35, 36). Treatments with either PD98059 (1 µM) or UO126 (1 µM) but not UO124 (1 µM), the negative control, were effective in abolishing the effect of SB202190 on p-p42/44MAPK (Fig. 3Go).



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  		Figure 2. Effect of a p38MAPK inhibitor on p42/44MAPK activation. A, Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated with (A) graded concentrations of SB202190 (SB+; 0.1 to 10 µM) or SB202474 (SB-; 10 µM) for 15 min and (B) SB+ (10 µM) for different time periods as indicated. The cells were then collected by centrifugation, dissolved in 1x sample buffer, and analyzed by Western blotting using a monoclonal antibody against p-p42/44MAPK as described in Materials and Methods. The blot presented is representative of three separate experiments. Blots were scanned and analyzed by Sigmagel. The relative densitometric readings [control (Con) = 1] from three separate experiments are presented in the bottom panel as mean ± SEM values (open columns, p-p44MAPK; hatched columns, p-p42MAPK). *, P < 0.05, significantly different from control.

 


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  		Figure 3. Effects of PD98059, UO126 and UO124 on SB202190-stimulated p42/44MAPK phosphorylation. Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated for 15 min with SB202190 (SB+; 10 µM) in the absence or presence of PD98059 (PD; 1 µM), UO126 (1 µM) or UO124 (1 µM). The cells were then collected by centrifugation, dissolved in 1x sample buffer and analyzed by Western blotting using a monoclonal antibody against p-p42/44MAPK as described in Materials and Methods. The blot presented is representative of three separate experiments.

 
Effect of p38MAPK inhibition on NE-stimulated p42/44MAPK activation
We have previously shown that in rat pinealocytes, p42/44MAPK can be activated by NE, the endogenous neurotransmitter (27). In the following experiment, we investigated the effect of p38MAPK inhibition on NE-stimulated p-p42/44MAPK. As shown in Fig. 4Go, NE alone caused a concentration-dependent increase in the level of p-p42/44MAPK. Cotreatment with NE and SB202190 resulted in a larger increase in p-p42/44MAPK than the response to NE or SB202190 alone, suggesting that the effect of NE and p38MAPK inhibition is additive. The time course of the response to NE and SB202190, alone and in combination, was also determined. As shown in Fig. 5Go, cotreatment with NE and SB202190 resulted in a sustained increase in p-p42/44MAPK that persisted for 3 h. In contrast, an increase in p-p42/44MAPK was not observed beyond 1 h when cells were treated with either NE or SB202190 alone. These results suggest that the presence of p38MAPK inhibition increases the magnitude as well as the duration of NE-activated p-p42/44MAPK.



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  		Figure 4. Interaction of SB202190 and NE in activating p42/44MAPK. A, Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated for 15 min with graded concentrations of NE (1 to 100 µM) in the presence or absence of SB202190 (SB+; 10 µM). The cells were then collected by centrifugation, dissolved in 1x sample buffer and analyzed by Western blotting using a monoclonal antibody against p-p42/44MAPK as described in Materials and Methods. The blot presented is representative of three separate experiments. B, Blots were scanned and analyzed by Sigmagel. The relative densitometric readings (Con = 1) from three separate experiments are presented as mean ± SEM values (top panel, p-p44MAPK; bottom panel, p-p42MAPK). * P < 0.05, significantly different from the corresponding treatment without SB+.

 


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  		Figure 5. Time-course of SB202190 and NE-induced p42/44MAPK activation. A, Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated for the indicated time with NE (10 µM), SB202190 (SB+; 10 µM) or both. The cells were then collected by centrifugation, dissolved in 1x sample buffer and analyzed by Western blotting using a monoclonal antibody against p-p42/44MAPK as described in Materials and Methods. The blots presented are representative of three separate experiments. B, Blots were scanned and analyzed by Sigmagel. The relative densitometric readings (Con = 1) from three separate experiments are presented as mean ± SEM values (top panel, p-p44MAPK; bottom panel, p-p42MAPK). * P < 0.05, significantly different from control.

 
Effects of p38MAPK inhibition on p-p42/44MAPK by pathways activated by NE in rat pinealocytes
To examine the interaction between p38MAPK inhibition and NE on p-p42/44MAPK, pharmacological agents were used to selectively stimulate individual signaling pathways that are known to be activated by NE in pinealocytes (37). Whereas activation of protein kinase A (PKA) by 1 mM of dibutyryl-cAMP caused a reduction in p-p42/44MAPK (Fig. 6AGo), activation of PKG (by 1 mM of 8-bromo-cGMP), PKC (by 0.1 µM of PMA) or elevation of intracellular Ca2+ (by 1 µM of ionomycin) was effective in elevating the level of p-p42/44MAPK (Fig. 6BGo). Cotreatment with SB202190 further enhanced the levels of p-p42/44MAPK maximally stimulated by 8-bromo-cGMP, PMA, or ionomycin, (Fig. 6BGo). In contrast, activation of PKA by dibutyryl-cAMP reduced SB202190-induced increase in p-p42/44MAPK (Fig. 6AGo). Inhibitors of cGMP-dependent protein kinase (KT5823; 1 µM), cAMP-dependent protein kinase (H89; 1 µM), PKC (calphostin C; 1 µM) and Ca2+/calmodulin-dependent kinase (KN93; 1 µM) also had no effect on the SB202190-induced increase in p-p42/44MAPK (Fig. 6CGo). Treatment with SB202190 (10 µM) did not have an effect on cAMP and cGMP levels (Table 1Go). These results suggest that SB202190 is not using the same pathway as NE in stimulating p42/44MAPK phosphorylation.



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  		Figure 6. Effects of protein kinase activators and inhibitors on SB202190-stimulated p42/44MAPK phosphorylation. Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated for 15 min with (A) dibutyryl-cAMP (DB-cA; 1 mM); (B) PMA (0.1 µM); 8-bromo-cGMP (Br-cG: 1 mM) or ionomycin (ION; 1 µM) in the presence or absence of SB202190 (SB+; 10 µM); or (C) SB202190 (SB+; 10 µM) in the absence or presence of KT5823 (1 µM), calphostin C (Cal-C, 1 µM), H89 (1 µM) or KN93 (1 µM). The cells were then collected by centrifugation, dissolved in 1x sample buffer and analyzed by Western blotting using a monoclonal antibody against p-p42/44MAPK as described in Materials and Methods. The blots presented are representative of four separate experiments.

 

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  		Table 1. Effect of SB202190 on cAMP and cGMP levels

 
Effect of phosphatase inhibitors on SB202190-induced increase in p-p42/44MAPK
One potential mechanism through which SB202190 can increase the level of p-p42/44MAPK is by inhibiting dephosphorylation of p42/44MAPK or components of the signaling cascade that activate p42/44MAPK. To test this possibility, two phosphatase inhibitors, calyculin A (a serine/threonine phosphatase inhibitor) and dephostatin (a protein tyrosine phosphatase inhibitor), were used (38, 39). Treatment with calyculin A (0.3 µM) or dephostatin (100 µM) alone elevated the level of p-p42/44MAPK. Under this condition, SB202190 (10 µM) remained effective in increasing the level of p-p42/44MAPK (Fig. 7Go). These results suggest that the increase in p-p42/44MAPK by SB202190 is probably through a mechanism that is independent of dephostatin- or calyculin A-sensitive phosphatases.



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  		Figure 7. Effects of protein phosphatase inhibitors on SB202190-stimulated p42/44MAPK phosphorylation. Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated for 15 min with calyculin A (Caly-A; 0.3 µM) or dephostatin (Dephos; 100 µM) in the absence or presence of SB202190 (SB+; 10 µM). The cells were then collected by centrifugation, dissolved in 1x sample buffer and analyzed by Western blotting using a monoclonal antibody against p-p42/44MAPK as described in Materials and Methods. The blot presented is representative of three separate experiments.

 
Effect of p42/44MAPK inhibition on p-p38MAPK
To further examine the interaction between p42/44MAPK and p38MAPK, the effect of p42/44MAPK inhibition on p38MAPK phosphorylation was determined. Treatment with two MEK inhibitors, PD98059 (1 µM) or UO126 (1 µM) (35, 36) reduced p-p42/44MAPK but had no effect on basal p-p38MAPK level (Fig. 8Go). In the same experiment, treatment with SB202190 (10 µM) was effective in elevating p-p42/44MAPK but had little effect on p-p38MAPK (Fig. 8Go). None of these treatments had an effect on the protein levels of p42/44MAPK and p38MAPK (Fig. 8Go). These results suggest that, whereas inhibition of p38MAPK phosphorylation could increase p42/44MAPK phosphorylation, inhibition of p42/44MAPK has no effect on p38MAPK activation.



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  		Figure 8. Effects of different MAPK inhibitors on p38MAPK and p42/44MAPK phosphorylation. Pinealocytes (1 x 105 cells/0.5 ml) were cultured for 24 h and treated for 20 min with SB202190 (SB+; 10 µM), PD98059 (PD; 1 µM) or UO126 (1 µM). The cells were then collected by centrifugation, dissolved in 1x sample buffer. Aliquots from the same samples were analyzed for p-p38MAPK and p-p42/44MAPK levels by Western blotting using monoclonal antibodies and p38MAPK and p42/44MAPK using polyclonal antibodies as described in Materials and Methods. The blots presented are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although p42/44MAPK and p38MAPK belong to two subfamilies of MAPKs and are activated by different MEKs (15, 16, 17), results from the present study indicate that there is a close relationship between these two subfamilies of MAPKs in rat pinealocytes. This is based on the observations that both basal and adrenergic-activated p42/44MAPK phosphorylation is significantly modulated by p38MAPK activities. Whereas inhibition of p38MAPK alone by specific inhibitors, SB202190 or SB203580, is effective in elevating the level of p-p42/44MAPK, inhibition of the p42/44MAPK activation by PD98059 or UO126 has no effect on p38MAPK phosphorylation. Taken together, these results suggest the presence of a signal input from p38MAPK that negatively regulates p42/44MAPK in rat pinealocytes. Furthermore, because the effect of SB202190 can be abolished by PD98059 and UO126, two MEK inhibitors, the site of action for the p38MAPK inhibitors is either at and/or upstream of MEK1/2.

We have previously shown that, in rat pinealocytes, NE, the endogenous neurotransmitter, can activate p42/44MAPK (27). In the present study, we found that inhibition of p38MAPK by SB202190 further enhances the NE-stimulated p42/44MAPK activation. However, neither the cGMP-PKG nor the cAMP-PKA pathway appears to mediate the effect of p38MAPK inhibition on p42/44MAPK activation. This is based on the observations that, whereas KT5823 is effective in abolishing the effect of dibutyryl cGMP on MAPK phosphorylation (27), neither H89, a PKA inhibitor, nor KT5823, a PKG inhibitor, has an effect on the increase in p42/44MAPK phosphorylation induced by the p38MAPK inhibitor. SB202190 also has no effect on cAMP and cGMP levels. Furthermore, the effects of maximal activation of PKA and PKG by pharmacological means on p42/44MAPK phosphorylation are additive to that of the p38MAPK inhibitor with PKA reducing and PKG further enhancing p42/44MAPK phosphorylation. Thus, it appears that the mechanism that mediates the effect of p38MAPK inhibition on p42/44MAPK phosphorylation is distinct from the known pathways used by NE in activating p42/44MAPK. However, the consequence of the additive effect of SB202190 on NE-stimulated p42/44MAPK phosphorylation is that the magnitude and duration of p42/44MAPK phosphorylation is significantly higher than that stimulated by NE or by p38MAPK inhibition alone. These results suggest that through inhibition of p38MAPK, the p42/44MAPK response is amplified. Moreover, interaction between p38MAPK and p42/44MAPK likely provides a mechanism through which input signals that activate different members of the MAPK families can be integrated.

The involvement of PKC in the activation of p42/44MAPK in other tissues is well established (17, 18, 20). In this study, we found that pharmacological activation of PKC by PMA is an effective means of activating p42/44MAPK in this tissue. However, our results argue against PKC in mediating the effect of p38MAPK inhibition on p42/44MAPK phosphorylation in rat pinealocytes. This is based on the observations that the effect of maximal activation of PKC by PMA on p42/44MAPK phosphorylation is additive to that of the p38MAPK inhibitor. Furthermore, calphostin C, a PKC inhibitor, has no effect on p42/44MAPK activation induced by the p38MAPK inhibitor. In contrast, calphostin C is effective in reducing the PMA-stimulated p42/44MAPK phosphorylation (our unpublished observation).

Another possible mechanism through which p38MAPK inhibitor can enhance p42/44MAPK phosphorylation is by inhibition of phosphatases that participate either directly or indirectly in regulating p42/44MAPK phosphorylation. Apart from MKP-1, protein phosphatase 2A has also been shown to be directly involved in the dephosphorylation of p-p42/44MAPK (40). Even though inhibition of serine/threonine phosphatases by calyculin A or tyrosine phosphatases by dephostatin are both effective means in activating p42/44MAPK in rat pinealocytes, neither phosphatase inhibitors appear to be involved in the effect of p38MAPK inhibition on p42/44MAPK phosphorylation. This is based on the observation that the p38MAPK inhibitor remains effective in causing a further increase in p-p42/44MAPK induced by the phosphatase inhibitors. However, these results could not exclude the involvement of phosphatases that are not sensitive to calyculin A or dephostatin in mediating the effect of the p38MAPK inhibitors.

Our results support that p42/44MAPK likely represents an important target through which multiple signal inputs are integrated in the rat pineal gland. In addition to its activation by NE through a PKG-dependent mechanism (27), we now found that NE activation of p42/44MAPK is also modulated by the state of p38MAPK activation. Whereas inhibition of p38MAPK causes an increase in p42/44MAPK phosphorylation, inhibition of p42/44MAPK has no effect on p38MAPK phosphorylation. These results suggest the presence of a one-way cross-talk between p38MAPK and p42/44MAPK in the rat pineal gland. Similar one-way cross-talk between p38MAPK and p42/44MAPK has previously been described in a rat hepatoma cell line (26) and activation of p38MAPK causes inhibition of p42/44MAPK has also been reported in other cell types (23, 25).

Although the function of p42/44MAPK in rat pinealocytes is not known, these kinases may be involved in gene transcription in the rat pineal gland because they play an important role in the regulation of nuclear transcription factor in other cells (41). The potential involvement p42/44MAPK in pineal gene transcription underscores the importance to determine the mechanism through which p38MAPK inhibition causes activation of MEK1/2 and p42/44MAPK. Elucidating the mechanism involved in the interaction between different members of the MAPK family will undoubtedly advance our knowledge on pineal cell biology. The importance of this line of research is also suggested by the recent observation that MAPK is involved in clock oscillation of the chick pineal gland (42).


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada. Back

Received June 21, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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