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Unidad de Endocrinología Molecular, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas, Madrid, Spain
(Requests for offprints should be addressed to M-J Obregón, Instituto Investigaciones Biomédicas, CSIC-UAM, Arturo Duperier 4, 28029 Madrid, Spain; Email: mjobregon{at}iib.uam.es)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsDetermination of D2 activityResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsDetermination of D2 activityResultsDiscussionReferences |
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The cDNAs coding for the three deiodinases have been isolated from rat, human and other species (Berry et al. 1991, Croteau et al. 1995, 1996). All of them contain one in-frame TGA codon that is translated as selenocysteine due to the presence of a specific structure, the selenocysteine insertion sequence (SECIS), in the 3'-untranslated region of their mRNAs (Berry et al. 1991, Davey et al. 1995).
In BAT, the best stimulation of D2 activity is obtained under cold exposure, mediated by the norepinephrine (NE) released from the sympathetic nerve endings (Silva & Larsen 1983). D2 produces most of the T3 found in BAT (Silva & Larsen 1985), which saturates the nuclear T3 receptors under cold exposure (Bianco & Silva 1987), and the T3 produced is necessary for the complete thermogenic function of BAT (Carvalho et al. 1991), namely, the full expression of the uncoupling protein (UCP-1), a specific marker of BAT. In floating brown adipocytes, D2 is stimulated by adrenergic agents (Obregón et al. 1987), and synergism between the 
1 and ß adrenergic pathways has been described (Raasmaja & Larsen 1989).
In addition, BAT D2 activity is upregulated by insulin, as shown after insulin injection to diabetic rats and by low D2 activity in BAT of diabetic rats (Silva & Larsen 1986b). In floating brown adipocytes, insulin stimulates D2 activity by increasing its Vmax (Mills et al. 1987).
Insulin is a key hormone for adipocytes, required for the increase in lipogenic enzymes associated with the differentiation of adipocytes. In brown adipocytes, lipogenesis and thermogenesis seem to have opposite roles, as dedifferentiation of the brown adipocytes is accompanied by increased lipid accumulation, acquisition of the white adipocyte phenotype and loss of the thermogenic marker UCP-1. On the other hand, during active thermogenesis, the lipids are the substrate used for mitochondrial combustion; therefore, lipogenesis is required for the thermogenic activity of BAT. Thus, both processes, thermogenesis and lipogenesis, are complementary, and T3 induces the expression of both UCP-1 and lipogenic enzymes (Bianco et al. 1992, García-Jiménez et al. 1993, Guerra et al. 1996, Hernández & Obregón 2000).
The effect of insulin on the deiodinases has also been studied. Insulin upregulates hepatic T3 production, as has been described in ‘low T3 syndromes’. In insulin deprivation (as in diabetes and fasting), T3 production is low due to low hepatic D1 deiodinase activity and mRNA (O’Mara et al. 1993), leading to low serum T3 levels. D2 activity in BAT is also low in these situations, reducing T3 production, T3-induced thermogenesis and hence the capacity to increase energy expenditure in fasting or diabetes. BAT D2 has been proposed as a source of circulating T3 (Fernandez et al. 1987), besides providing the T3 required for UCP-1 increases.
Using primary cultures of rat brown adipocytes, we previously showed that in hypothyroid conditions NE poorly stimulates D2 activity and that T3 is required for and amplifies the adrenergic D2 response by 10–20-fold. This effect of T3 is observed with NE or ß3 adrenergic agents (Hernández & Obregón 1996). The stimulatory effect of T3 on the adrenergic response of D2 requires de novo protein synthesis and is inhibited by actinomycin. Recently, we have shown that T3 is an absolute requirement for the adrenergic stimulation of D2 mRNA and that T3 itself increases D2 mRNA (Martinez-deMena et al. 2002).
In the present paper, we studied the regulation of the adrenergic stimulation of D2 by insulin in primary cultures of rat brown preadipocytes. We show that insulin plays a fundamental role, upregulating the adrenergic stimulation of D2 activity and mRNA expression in brown adipocytes. The signaling pathways and mechanisms involved in the insulin action, ERK 1/2 and PI3K, are also examined. We conclude that insulin and the stimulation of the ERK 1/2 pathway are essential for the D2-mediated production of T3 in brown adipocytes.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsDetermination of D2 activityResultsDiscussionReferences |
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The source of most of the reagents used has been previously described (Hernández & Obregón 1996). We purchased newborn calf serum (NCS) from Flow (Paisley, UK) or from Gibco Life Technologies (Uxbridge, UK); collagenase, bovine insulin, ascorbic acid, dithiothreitol (DTT), 6-n-propyl-2-thiouracil (PTU), NE and T3 from Sigma; insulin-like growth factor (IGF)-I and IGF-II from Boehringer Mannheim (Mannheim, Germany); wortmannin, PD-98059 (PD), LY-294002 (LY) and the proteasome inhibitor MG132 from Calbiochem (Schwalbach, Germany); oligo(dT) cellulose from New England Biolabs (Beverly, MA, USA); p-ERK (MAP kinases, p44 and p42) antibodies and the Western blotting luminol reagent kit from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and Phospho-AKT (PKB) rabbit antibodies from PharMingen (Becton Dickinson).
Primary cultures of brown adipocytes
Precursor cells were obtained from the interscapular BAT of 20-day-old rats (Sprague-Dawley), isolated by the method described by Néchad et al.(1983), by collagenase digestion (0.2%) and filtration through 250 µm silk filters. Mature cells were allowed to float, and the infranatant was put through 25 µm silk filters and centrifuged. Precursor cells were seeded at a density of 1500–2000 cells/cm2 on day 1, and grown in DMEM supplemented with 10% NCS, 3 nM insulin, 10 mM HEPES, 50 IU penicillin and 50 µg streptomycin/ml, and 15 µM ascorbic acid. Culture medium was changed on day 1 and every second day thereafter. Precursor cells proliferate actively under these conditions, reach confluence at day 4 or 5 after seeding (40 000–60 000 cells/cm2) and differentiate into mature brown adipocytes. All studies and treatments were done in fully differentiated brown adipocytes (at days 8–9 after seeding).
Both NCS and hypothyroid serum were used for culture. Hypothyroid serum, obtained as previously described (Samuels et al. 1979), contained 10% or less of the original amount of thyroid hormones, as assessed by RIA (Morreale de Escobar et al. 1985). Total and free T4 and T3 in NCS and hypothyroid serum have been recently described (Martinez-deMena et al. 2002).
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Determination of D2 activity |
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TopAbstractIntroductionMaterials and methodsDetermination of D2 activityResultsDiscussionReferences |
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High specific activity 125I-T4 (>3000 µCi/µg) was obtained in our laboratory with chloramine T and T3 as substrate (Obregón et al. 1989, Hernández & Obregón 1996). Before each assay, 125I-T4 was purified by paper electrophoresis to separate the contaminating iodide. Assay blanks were always less than 1% of the total radioactivity. The validation of the assay has been published (Hernández & Obregón 1996).
RNA preparation and Northern blot analysis
For isolation of Poly(A)+ RNA, cells were harvested, and mRNA was isolated using oligo(dT) cellulose, as previously described (Vennström & Bisop 1982). For Northern analysis, Poly(A)+ (5 µg) was denatured and electrophoresed on a 2.2 M formaldehyde/1% agarose gel in 1 MOPS buffer and transferred to nylon membranes, as previously described (García-Jiménez et al. 1993, Hernández et al. 1998). A 1.988 kb fragment of a rat D2 cDNA clone (Croteau et al. 1996), including the entire coding region, was used as a probe by labeling with [-32P]dCTP, using random primers. Filters were hybridized with D2 cDNA and cyclophilin as a control to correct for differences between lanes in the amount of Poly(A)+ mRNA (Danielson et al. 1988). Autoradiograms were obtained from the filters and quantified by computer-assisted densitometry (Molecular Dynamics, Sunnyvale, CA, USA). All the experiments were repeated at least two times. The more complete or representative experiments are shown.
Western blotting
Total protein extracts (50 µg/well) were resolved by SDS–PAGE and transferred to PVDF transfer membranes (Biotechnology Systems, NEN Research Products, Boston, MA, USA), in a buffer containing 25 mM Tris, 200 mM glycine and 20% methanol. After blocking of the membrane with 5% low-fat dried milk in Tris-buffered saline containing 0.05% Tween-20, the levels of phosphorylated ERK (p44 and p42) were detected with a 1:5000 dilution of the specific antibody (pERK), followed by a 1:2000 dilution of goat antimouse IgG-HRP (Santa Cruz). Phospho-AKT was detected with a 1:1000 dilution of a specific rabbit antibody (anti-pAKT) from Pharmingen (Becton Dickinson), followed by a 1:5000 dilution of goat antirabbit immunoglobulin (Ig) G (Santa Cruz). Immunoreactive bands were visualized by enhanced chemiluminescence (Western blotting luminol reagent, Santa Cruz).
Statistical analysis
Mean values ± S.E. are given. When required, one-way analysis of variance was done, after homogeneity of variance was ensured by square root or logarithmic transformations, if not found with the raw data. The significance of differences between groups was assessed by the protected least-significant-difference test. All the calculations were done as described by Snedecor and Cochran (1980).
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Results |
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TopAbstractIntroductionMaterials and methodsDetermination of D2 activityResultsDiscussionReferences |
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Brown adipocytes grown in the presence of insulin had many lipid droplets and round shapes, while no lipid accumulation was observed in the insulin-depleted cells.
Insulin upregulates D2 activity in floating rat brown adipocytes (Mills et al. 1987). However, in cultured rat brown adipocytes, we did not observe increases in D2 activity after addition of human or bovine insulin (from 3 to 3000 nM for 2–6 h) when it was added alone (without NE and T3) (not shown). But, as insulin improves the adrenergic stimulation of D2, all the experiments were done in the presence of NE plus T3, in conditions previously tested (Hernández & Obregón 1996, Martinez-deMena et al. 2002).
Figure 1
shows that the adrenergic stimulation of D2 activity improved in the presence of insulin. Figure 1A shows the time-course response of D2 to NE in the presence or absence of insulin. D2 reached a peak 8 h after addition of NE, and the adrenergic response was much lower in the absence of insulin. The effect of insulin depletion for a short and a long time was also examined. Insulin depletion for 1 day (Fig. 1B) decreased D2 activity by 35%, and D2 was fully recovered after a brief exposure to insulin (6 h). Prolonged insulin depletion (3 days; Fig. 1C) caused a 50% reduction in D2 activity. Addition of insulin during the last 6 h had no effect, but D2 activity was partially restored 24 h after addition of insulin. Therefore, the adrenergic stimulation of D2 is upregulated in the presence of insulin, and the recovery of D2 activity is dependent on the insulin-deprivation status of the adipocytes. The maximal inhibition of D2 was observed after 2–3 days of insulin depletion (30–50% of control values), and the inhibition was maintained thereafter; insulin replacement had a clear effect at 24 h.
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) did not overcome the decreases in D2 activity observed after insulin deprivation.
To test whether the insulin effect was mediated through the IGF receptors, we investigated the effect of increasing doses of insulin, IGF-I and IGF-II on the adrenergic stimulation of D2 activity. Figure 2 shows that insulin increases the adrenergically stimulated D2 activity at very low doses (twofold at 0.03 nM), in a dose-dependent way, and this effect is reproduced only at much higher doses of IGF-I or IGF-II. Maximal stimulation of D2 activity is already obtained with physiologic doses of insulin (3 nM). Insulin is more potent than IGFs, suggesting that its effects are not mediated through the IGFs receptors.
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The actions of insulin are mediated through the binding to its specific membrane receptors that, by activating the insulin receptor tyrosine kinase, leads to the phosphorylation of several substrates, mainly IRS-1, which acts as the anchor protein activating several pathways as PI-3 kinase (PI3K), and also results in the sequential activation of the Ras, Raf and MAPK cascade.
We first tested whether the ERK 1/2 signaling pathway is implicated in the regulation of D2 by insulin. Confluent preadipocytes were depleted from insulin for 4 days. Insulin, added during the last 24 h (Fig. 3), partially restored D2 activity (P < 0.05). Addition of PD resulted in inhibition of the insulin effect, indicating that ERK 1/2 is involved in the regulation of the adrenergic stimulation of D2 activity by insulin. We also tested whether PI3K is involved in the signaling pathway by adding two inhibitors of PI-3 kinase, wortmannin (W) and LY-294002 (LY), the latter reported to be more specific and stable. Addition of 20 nM W did not suppress the insulin effect (even after repeated additions, possibly due to its instability in aqueous medium (results not shown), while LY inhibited D2 with an effect similar to PD, indicating that both pathways (ERK 1/2 and PI3K) are involved in the activation of D2 by insulin.
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) or absence of serum (Fig. 4B). Cells were always stimulated with NE and T3. In the presence of 10% serum (Fig. 4A), insulin depletion decreased D2 activity by 25%, and addition of PD or LY decreased D2 activity, in both the presence and the absence of insulin, indicating that both pathways are possibly stimulated by serum in insulin-depleted cells. Using serum-free medium (Fig. 4B), the adrenergic stimulation of D2 was observed only when insulin was present, but was very low in the absence of insulin and serum, indicating that ERK 1/2 and PI3K are required for the adrenergic stimulation of D2 activity. The addition of LY plus PD did not induce a stronger inhibition (not shown). We conclude that the adrenergic stimulation of D2 by NE and T3 is not enough by itself to increase D2 activity and requires the presence of an ERK 1/2 or PI3K activity induced by insulin and/or serum. Interestingly, the basal levels in the presence of T3 increased in the absence of insulin plus serum, suggesting that both agents inhibit the basal D2 activity in T3-treated cells.
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We further checked the activation of ERK 1/2 and PI3K by Western blots under the three stimuli used: insulin, serum and NE. Figure 5A shows that all three stimulate ERK 1/2 (p42 and p44), and serum was more potent than insulin and NE. All three stimuli were inhibited by PD, although the inhibition of serum was not complete. The effect of NE is dose and time dependent (maximum at 10 min and at 0.5–2 µM NE) (results not shown). Figure 5B shows that T3 alone has no effect on ERK 1/2, while the addition of insulin to NE plus T3 increases ERK 1/2 activity, showing an additive effect between the signaling pathways of NE and insulin.
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shows that insulin increases pAKT, which is inhibited by LY, but not by PD. The addition of LY plus PD resulted in complete inhibition of pAKT. The addition of serum also induced pAKT, which was slightly inhibited by LY.
We also tested the effect of NE on pAKT. NE by itself did not induce pAKT (Fig. 5D), but enhanced the effect of insulin (lane 5) or insulin plus serum (lane 7), suggesting an interaction between NE and insulin.
Effect of insulin on the adrenergic stimulation of D2 mRNA expression
We tested whether the effects of insulin observed on D2 activity were also observed at the mRNA level. Different results were obtained when cells were depleted of insulin for a long time, around cellular confluence (from day 4, for 5 days) or for a shorter time, during the differentiation period (from day 6, for 3 days). When insulin was suppressed very early, D2 mRNA expression decreased by 50% (Fig. 6A) in agreement with the findings for D2 activity. The addition of physiologic doses of insulin or IGF-I for 24 h increased D2 mRNA expression by 4.5- and 2.7-fold respectively above control values. However, when insulin was withdrawn for a shorter time and during the differentiation period (at day 6, Fig. 6B), we found small variations and the addition of insulin or IGF-I led to small increases (n.s.) in D2 mRNA expression. This experiment was repeated several times, and the mean values are represented in Fig. 6A and B. Therefore, D2 mRNA decreases only after prolonged insulin depletion, in contrast to the changes observed in D2 activity.
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) are not caused by stabilization of the mRNA half-life, because we tested the disappearance of D2 mRNA during 5 h in the presence of actinomycin D, and no difference was observed between the presence or absence of insulin (results not shown). PD decreased D2 mRNA stimulated by insulin or serum (results not shown), confirming the ERK 1/2 dependency of the adrenergic stimulation of D2 also at the transcriptional level. We did not observe the inhibitory effect when using LY.
The action of actinomycin D and cycloheximide (CHX) previously studied in control cells (Martinez-deMena et al. 2002) was tested in cells depleted of insulin at days 4 and 6, and we observed similar results in all three situations. Actinomycin D decreases D2 mRNA levels to undetectable levels, and CHX leads to accumulation of D2 mRNA in all three cases (Fig. 7A). These results suggest that insulin acts as a modulator of the transcriptional increases due to adrenergic stimulation of D2. When T3 was used alone (Fig. 7B) (T3 for 72 h without adrenergic stimulation), as previously reported (Martinez-deMena et al. 2002), insulin depletion led to complete loss of the D2 mRNA signal, and actinomycin and CHX had the same effect as described with NE plus T3. These data suggest that insulin is required for stimulation by T3 of D2 mRNA.
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). Higher D2 expression was found near cellular confluence. In fact, D2 mRNA decreases during differentiation (results not shown), showing that a delayed differentiation state could not cause the D2 mRNA decreases in insulin-depleted cells. The capacity of the cells to respond to adrenergic stimulation of D2 mRNA decreases along with differentiation.
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As we did not observe a direct action of insulin by itself when it was added to the cells, we investigated whether the depletion of insulin could decrease D2 activity half-life and modify its degradation pathways. We analyzed D2 activity half-life in the presence or absence of insulin; in the latter, we did not find a higher turnover of D2 activity (Fig. 9). In some experiments, the absence of insulin induced a slight stabilization of D2 activity. Therefore, the decreases observed in the absence of insulin are not due to a higher turnover rate of D2 activity.
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, left panel). D2 increases (180%) were observed with 10 µM MG132 for 1–2 h. As previously reported (Steinsapir et al. 1998), MG132 is also able to reverse the inhibition of D2 activity by CHX (Fig. 10, right panel). Finally, we compared the effect of MG132, added during the last hour of adrenergic stimulation, with or without insulin (Table 1). The use of MG132 led to increases in D2 activities (166%). In the absence of insulin, a smaller increase was observed (138%). This excludes a higher D2 turnover in the absence of insulin, as less D2 is degraded in this situation, in terms of both percentage and absolute units. It also indicates that insulin does not inhibit proteasome activity, because we would have found higher degradation rates in the absence of insulin.
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Discussion |
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TopAbstractIntroductionMaterials and methodsDetermination of D2 activityResultsDiscussionReferences |
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Previous reports found large increases of D2 activity after insulin injections to rats (Silva & Larsen 1986b) or a progressive increase in Vmax when insulin was added to floating brown adipocytes (Mills et al. 1987). In the present study, we found a clearly positive effect of insulin on D2 activity and mRNA, once adrenergically stimulated, but we did not find an effect of insulin by itself, as described in rats, implying that the in vivo effect could be mediated through other pathways (Silva & Larsen 1986b). Insulin stimulates the Vmax of D2 activity in floating brown adipocytes, as shown with higher insulin dosages (3000 µU/ml=25 nM). Our study used lower insulin concentrations (3 nM), but we cannot exclude that floating adipocytes are more mature and respond better to insulin than cultured adipocytes. It has been suggested that the changes in Vmax are due to an increase in D2 protein synthesis (Mills et al. 1987), but the decrease of D2 degradation or the stabilization of D2 half-life that could also lead to accumulation of D2 was not examined.
We observed that adrenergically stimulated D2 activity decreases in the absence of insulin and is induced at very low concentrations (0.05 nM), maximal increases being obtained at physiologic doses (3 nM). Insulin acts through its own receptors, not through IGF receptors, and the recovery of the D2 response depends on the length of insulin depletion. Our results also indicate that the adrenergic stimulation, though lower, is maintained even in the absolute absence of insulin, but in this case the presence of serum is required.
The experiments done to delineate the signaling pathway of insulin on D2 activity indicate that insulin acts through both signaling pathways, ERK 1/2 and PI-3 kinase. Most interesting is the fact that the activation of ERK 1/2 by insulin and/or serum is required for the adrenergic stimulation of D2 activity and mRNA. This new finding points to the interrelationship between different signaling pathways and agrees with the report that NE activates ERK 1/2 MAPK (Shimizu et al. 1997). We further show an increase in the phosphorylation of ERK 1/2 MAPK when insulin and NE are added together (Fig. 5B). It has also been reported that ß3 adrenergic stimulation activates p38 MAPK in murine brown adipocytes, and this pathway seems to be required for this stimulation (Cao et al. 2001). In rat pinealocytes, NE stimulates the phosphorylation of p42/44 MAPK, which in turn is modulated by the activation state of p38 MAPK (Man et al. 2004). The link between these two signaling pathways (MAPKs and CRE) becomes more important and involves routes different from the simple stimulation of the CRE. The fact that D2 is stimulated by fibroblast growth factors in astroglial cells (Courtin et al. 1990) also supports the role of ERK 1/2 signaling pathways in the stimulation of D2. D2 increases in situations in which recruitment of BAT is found, together with an increased proliferation rate (cold exposure and hypothyroidism), indicating a possible role for the relationship between D2 activities and MAPK signaling pathways. The other pathway explored, PI3K, stimulated by insulin and serum, but not by NE, is also enhanced by the combined presence of NE, insulin and serum.
The mechanisms of the insulin action on D2 are not clear. Insulin could act at different levels, as by increasing the transcriptional effect of NE. Insulin has been reported to stimulate both CREB phosphorylation and transcriptional activation in HepG2 and 3T3-L1 cells, through serine 133, the PKA site, and this process is blocked by PD98059 (Klemm et al. 1998). Our results confirm the positive effect of insulin on the transcriptional activation of D2 by NE and disclose a synergy between the PKA and ERK 1/2 pathways, a novel aspect of the adrenergic regulation of D2 in brown adipocytes. The CRE is present in the human and rodent proximal D2 promoter, and seems to play an important role in the full D2 expression. The functional interaction of insulin with CREB remains to be proven for D2. A putative serum response element (SRE) is found in the rat D2 promoter.
Insulin could also act at the post-transcriptional level, modulating D2 half-life or the activity of proteasome (degradation pathways); finally, it could act by promoting specific differentiation aspects necessary for D2 expression. We have analyzed several of these aspects. Our results exclude a shorter D2 half-life or a higher turnover rate of D2 activity as the cause of the low D2 values found in the absence of insulin. The inactivation of D2 activity has been reported to occur through the proteasome-dependent pathway (Steinsapir et al. 1998). Our study of the proteasome activity confirms the role of this pathway in D2 degradation and excludes that the absence of insulin results in a higher degradation of D2. The role of insulin as an inhibitor of proteasomal activity (Bennett et al. 2000) is not con-firmed in our cells, pointing to pretranslational events.
Our studies at the mRNA level indicate that the adrenergically increased D2 mRNA is rather stable and decreases only after prolonged insulin depletion or when it starts at very early stages (before confluence). These decreases are not observed in shorter insulin depletion (3 days), and small increases (n.s.) in D2 mRNA are even observed (Figs 6 and 7). These increases are not due to accumulation of D2 mRNA, as D2 mRNA half-life does not change in the absence of insulin. Therefore, the decreases in D2 activity due to the absence of insulin are found at the mRNA level, only after prolonged insulin depletion. Another possible explanation is that the early depletion of insulin delays the switch to the differentiation program of the adipocytes, which occurs around cellular confluence (Bronnikov et al. 1999). But this is not the case, as higher D2 mRNA expression is found around confluence. The response to insulin is larger in this situation (Fig. 6A), a fact not observed once the adipocytes are already differentiated (Fig. 6B).
The experiments performed with inhibitors of transcription or protein synthesis gave similar results to those reported in control cells under adrenergic stimulation (Fig. 7), suggesting that insulin modulates the transcriptional increases due to adrenergic stimulation.
From Fig. 4, it is obvious that T3 itself has a very small effect in increasing basal D2 activity in brown adipocytes and its action is inhibited by insulin and serum, in contrast with the T3 effects on D2 mRNA, where insulin is required for induction of D2 mRNA after prolonged exposure to T3.
D2 is an enzyme highly regulated at both the transcriptional and post-transcriptional levels. The present report studies most of the modulators of D2 activity in brown adipocytes: adrenergic stimulation (NE), thyroid hormones (T3), insulin and growth factors (serum) and the interaction of their signaling pathways. This emphasizes the tight regulation of T3 production in brown adipocytes and its importance for thermogenesis. D2 has been recently found to be very low in C/EBP-null mice, together with low BAT T3 and impairment of many genes involved in thermogenesis (UCP-1 and transcription factors) (Carmona et al. 2002). This implies that BAT D2 is linked to the differentiation program of brown adipocytes, which is highly dependent on C/EBP, and D2 is possibly required for the full achievement of the thermogenic capacity. It also indicates that D2 seems to be a target of C/EBP and that its functionality depends on the full activity of these transcription factors.
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and methodsDetermination of D2 activityResultsDiscussionReferences |
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Received 25 August 2004
Accepted 7 September 2004
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