Differential Regulation of Immediate Early Gene ...

Biochemical and Biophysical Research Communications 265, 664 – 668 (1999) Article ID bbrc.1999.1734, available online at http://www.idealibrary.com on

Differential Regulation of Immediate Early Gene Expression in Preadipocyte Cells through Multiple Signaling Pathways Hiroyuki Inuzuka,* ,† ,1 Rika Nanbu-Wakao,* ,1 Yasuhiko Masuho,* Masa-aki Muramatsu, 1 Hideaki Tojo,† and Hiroshi Wakao* ,2 *Helix Research Institute, 1532-3 Yana, Kisarazu-shi, Chiba, 292-0812, Japan; and †Department of Applied Genetics, Institute of Animal Resource Science, Graduate School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyou-ku, Tokyo 113, Japan

Received October 8, 1999

Using digoxigenin (DIG)-based differential hybridization, a series of immediate early genes (IEG) was identified following the adipogenic stimulation in 3T3-L1 preadipocyte cells. Most of the known IEGs were identified as well as new members such as zf 9 and Stra13. To delineate possible signaling pathways accounting for these gene expression, a subset of specific kinase inhibitors, SB203580, PD98059, rapamycin, LY294002, and Ro-32-0432, which inhibit p38 (HOG), MEK (MAPKK), S6 kinase, PI3 kinase, and protein kinase C (PKC), respectively, were employed. The IEGs were classified into three categories according to their susceptibility to the inhibitors. Expression of the first group (c-fos, jun-B, egr-1, tis11, tis21, thrombospondin-1, erp, thyroid hormone receptor [N-10], cyr61, and zf 9) was mainly dependent on PKC and MEK pathways, while that of the second class (gene33 and tis10) exhibited an additional dependence on PI3 kinase pathways. The third one (Id-3, gly96, and Stra13) was characterized in that none of these inhibitors interfered with gene expression. Our results suggest that the induction of IEGs by the adipogenic stimuli is mediated by common as well as distinct signaling pathways. © 1999 Academic Press

Extracellular stimuli regulate a spectrum of cellular events such as cell growth, differentiation and death by altering the gene expression profile. These include induction of previously quiescent genes or repression of active genes. Resulting induction of immediate early genes (IEGs) then triggers transcriptional cascades, which ultimately lead to the different biological phenotypes. Thus, identification and characterization of 1

These authors equally contributed to this work. To whom correspondence should be addressed. Fax: 81-438-523952. E-mail: [email protected]. 2

0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

IEGs is essential for understanding molecular mechanisms underlying these metabolic changes in cells. Furthermore, the mechanisms that mediate rapid gene activation have been a matter of interest. Although much efforts have been devoted to identify IEGs and to elucidate possible induction mechanisms, these studies are largely based on the experiments using only a single stimulus such as serum, peptide growth factors, or compounds that mimic the action of signaling molecules in cells [for example, see (1)]. We have been interested in the mechanisms by which diverse stimuli simultaneously and coordinately exert their effects on IEG expression in preadipocyte cells. Previous studies have demonstrated that c-fos, c-jun and jun B are induced as IEG in 3T3-L1 preadipocyte cells by tumor necrosis factor, 8-bromo-cAMP and 3-deazaadenosine (2– 4). However, little is known about the other IEGs. Fetal bovine serum (FBS), methlyisobutylxanthine (MIX), dexamethasone (DEX) and insulin are adipogenic hormones required for 3T3-L1 cells to differentiate into adipocytes. Upon treatment with these hormones, quiescent 3T3-L1 cells re-enter the cell cycle following clonal expansion. After subsequent proliferation cessation, they eventually change the morphology and express adipocyte specific marker genes such as adipsin, aP2, PPARg, and C/EBPa (5, 6). In this regard, identification and characterization of IEGs in 3T3-L1 cells should shed light on the molecular mechanism by which the IEGs trigger clonal expansion, growth arrest and finally adipocyte differentiation. Differential hybridization is one of the most classical method to detect genes expressing at different levels in two biological samples (7, 8). The method has been originally performed using the radiolabeled cDNA probes with either 32P or 33P. However, use of the radioactive probes has intrinsic problems such as limited sensitivity and short half-life of probes. To circum-

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vent these problems, we introduced non-radioactive labeling using the digoxigenin (DIG) nucleotide. DIGbased system is relatively simple and reliable method to detect DNA. In this report we demonstrate that most of the IEGs previously known to be induced by mitogens are identified as IEGs in 3T3-L1 cells by using DIG-labeled cDNA probes. In addition, two clones, zf9 and Stra13 were found to be IEGs. Further, we have delineated the signaling pathways which might contribute to the expression of these genes by using the specific kinase inhibitors. Our data disclose that IEG expression in preadipocyte cells is mediated by different as well as by common signaling pathways. MATERIALS AND METHODS Reagents. Bovine insulin, dexamethasone, rapamycin, LY294002 and cycloheximide were purchased from Sigma. Methylisobutylxanthine was from Wako Chemicals Co., Japan. SB203580 and Ro-320432 were obtained from Calbiochem. PD98059 was purchased from BIOMOL Research Lab. Cell culture and challenge with the adipogenic hormones. 3T3-L1 preadipocytes (NIHS cell bank, Japan, Catalogue Number JCRB9014) were maintained in growth medium of Dulbecco’s Modified Eagle Medium (DMEM; Nisseiken, Japan) containing 10% normal calf serum (CS; GIBCO BRL). For adipogenic stimulation, cells were grown to confluence and challenged with fresh DMEM containing 10% fetal bovine serum (FBS; GIBCO BRL), 1 mM DEX, 0.5 mM MIX, and 10 mg/ml of insulin for the indicated times. For the inhibitor experiments, indicated amount of the reagent was added to the medium one hour before treatment with the cocktail. Differential plaque hybridization. To isolate differentially expressed cDNA clones, we screened a lZipLox cDNA library (GibcoBRL) made from 3T3-L1 cells treated for 3 h with the adipogenic cocktail in the presence of 10 mg/ml of cycloheximide. The library was synthesized according to the instructions supplied by the manufacturer. Approximately, 1.6 3 10 6 pfu primary clones were obtained with an average insert size of more than 1.5 kbp. The cDNA library was plated at density about 1000 plaques per 10 cm plate and screened. Replicate nylon filters (Hybond-N1, Amersham-Pharmacia) were used for differential screening. The first filter was hybridized with cDNA probes prepared from untreated cells while the second filter was hybridized with those prepared from treated cells. Hybridization was carried out in the standard hybridization solution containing DIG-labeled probes (Boehringer Mannheim). DIG-labeled cDNA probes were prepared from uninduced and induced poly(A) 1RNA by incorporating DIG-11-dUTP according to the manufacturer’s protocol (Boehringer Mannheim) except for the use of Superscript II (GibcoBRL). Differentially hybridizing plaques were picked and purified through the second screening under the same hybridization conditions. The cDNA inserts from the purified clones were recovered in plasmid pZL1 (GibcoBRL) according to the manufacturer’s protocol and subjected to DNA sequencing (ABI 377 DNA sequencer). Northern blot analysis. Total RNA was isolated according to the method of acid-guanidine-phenol-chloroform (9). Indicated amount of the total RNA was fractionated on 1% agarose/2.2 M formaldehyde gel electrophoresis and transferred to a positively charged nylon membrane (Boehringer Mannheim). rRNA was stained on the filters with methylene blue to assess RNA loading and transfer efficiency (10). The DIG-labeled antisense RNA probes were transcribed from EcoRI-linearized cDNA plasmid pZL1 and Northern blot analysis was performed according to the protocol provided by the supplier (Boehringer Mannheim).

TABLE 1

Summary of the Immediate Early Genes Induced by Adipogenic Hormones Identity

Frequency (number of clones)

N-10 (thyroid hormone receptor) egr-1 (tis 8) tis21 erp (dual specific MAPK phosphatase) thrombospondin-1 (TSP-1) tis11 cyr 61 Id-3 gene 33 gly 96 pip 92 jun-B c-fos tis10 (cox-2) metallothionein-1 Stra 13 zf 9

20 5 5 4 3 3 2 2 2 2 2 2 2 1 1 1 1

RESULTS AND DISCUSSION Identification of Immediate Early Genes by Differential Hybridization Using DIG-Labeled Probes To isolate genes induced or suppressed by the cocktail of FBS, MIX, DEX and insulin, we have prepared a primary cDNA library from 3T3-L1 cells treated with these cocktails for three hours in the presence of cycloheximide. DIG-labeled first strand cDNAs were constructed from both treated and untreated cells. Phage plaques lifted onto a membrane in duplicate were hybridized with each DIG-labeled cDNA. Clones that appeared to be differentially expressed were subjected to the second screening and single clones were isolated. A total of 70.000 clones were screened and 150 candidate clones were sequenced. Sequence analysis revealed that more than half of these clones were rRNA (data not shown). Most of the other clones (total 59 clones) showed enhanced expression (see below), while few clones exhibited attenuated expression by Northern blot analysis (data not shown). As differentially expressed genes were isolated independently, statistic analysis was performed to evaluate the frequency of each gene. Table 1 shows the identity of differentially expressed genes and their redundancy. The most redundant clone was N-10 (thyroid hormone receptor) which appeared more than 20 times, suggesting that this clone is most abundant mRNA. The next frequent clones were egr-1 and tis21 that appeared 5 times. erp (dual specific MAPK phosphatase) was detected for 4 times, while Thrombospondin-1(TSP-1) and tis11 scored 3 times. jun-B, c-fos, Id-3, gly96, pip92, cyr61, and gene33 appeared twice. tis10, metellothionein-1,

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p19 cells (22). Our present data show that Stra13 gene is one of the IEGs in 3T3-L1 cells induced by the cocktail of FBS, MIX, DEX and insulin. Recently Stra13 has been identified as a potential IEG in NIH3T3 cells challenged with platelet derived growth factor (PDGF) using the oligonucleotide array, though the actual induction remains to be verified by Northern blot analysis (23). In light of these data, FBS which contains PDGF may account for the induction of this gene in 3T3-L1 cells. Like in the case for zf9, the target of this factor in 3T3-L1 cells remains to be elucidated. Diverse Signaling Pathways Are Responsible for the Induction of the Immediate Early Genes (IEGs)

FIG. 1. Northern blot analysis of the IEGs. An equal amount of the total RNA (4 mg/lane) isolated at the indicated time point with or without stimulation was probed with the antisense DIG-labeled riboprobes corresponding to jun-B, egr-1, tis10, tis11, N-10, cyr 61, erp, Id-3, gene 33, Stra13, and zf 9 gene.

zf9, and Stra13 were detected only once. It is noteworthy that most of the genes which have been previously reported to be IEG in NIH-3T3 or PC-12 cells was identified in 3T3-L1 preadipocyte cells using DIGlabeled cDNA probes (11–18). This indicates that DIGbased screening that we performed is quite efficient and reliable. The inducibility of each clone was verified by Northern blot analysis. As shown in Fig. 1, jun-B, egr-1 and tis10 were rapidly induced within an hour and thereafter their expressions dropped to undetectable level. Intriguingly, although serum induces tis10 in human fibroblast cells, the mRNA continues to accumulate up to 6 hr (19). The difference in these tis10 mRNA kinetics may lead to different biological output. Further study will be required to elucidate the biological significance of the different kinetics. N-10, cyr61, erp, Id-3 and gene33 were also rapidly induced, though the expression level of these genes peaked at one hour and the amount of these mRNA gradually decreased by 24 hr (lanes 1– 6). In contrast to these genes, mRNA for zf9 and Stra13 were readily present in the quiescent status and challenge with the stimuli enhanced both mRNA levels within an hour (Fig. 1, lanes 2). zf9 mRNA was then rapidly declined and a low level of expression was detected for 24 hr (Fig. 1, lanes 3– 6). Rat zf9 is induced in stellate cell during early hepatic fibrosis and transactivates promoters of collagen a1(I), TGFb1 and TGFb receptors, types I and II (20, 21). However, it is yet to be determined the role of zf9 in the early phase of preadipocyte cell differentiation. Stra13 mRNA also decreased to the prestimulated level after 3 hr, then gradually attenuated within 24 hr (Fig.1, lanes 3– 6). Stra13 has been identified as a basic helix-loop-helix protein induced by retinoic acid and its overexpression results in neuronal differentiation of

Having cloned a series of IEGs in 3T3-L1 cells, we then addressed a question as to how these gene expressions are regulated. We attempted to delineate which signaling pathways contribute to the expression of these genes using the specific kinase inhibitors. SB203580 and PD98059 which specifically inhibits p38 MAPK and MEK activity, respectively, were used to examine the involvement of the MAP kinase pathways. Rapamycin and LY294002 that inhibit S6 kinase and PI3 kinase activity, respectively, were employed to assess the contribution of PI3 kinase. Besides these inhibitors, Ro-32-0432 was used to analyze the effects of protein kinase C (PKC). According to the sensitivity to the each inhibitor, we have categorized these IEGs into three classes. One group exhibited a similar inhibition pattern where MEK and PKC pathways mainly contributed to the expression of the genes. These include c-fos, jun-B, egr-1, tis11, tis21, thrombospondin-1 (TSP-1), erp, thyroid hormone receptor (N-10), cyr61 and zf9 genes (Fig. 2A). Another class comprised gene33, tis10 where MEK, PKC and PI3 kinase pathways contributed to the gene expression (Fig. 2B). The third one composed of Stra13, gly96 were characterized in that none of the inhibitors significantly affected the expression (Fig. 2C). Previous studies have demonstrated that TPA induces IEGs called TIS (TPAinducible sequences) (24). Since Ro-32-0432 displays a selectivity for PKC a, PKC bI, and PKC e (25, 26), it is conceivable that these PKC isoforms are responsible for the expression of TIS (tis8 [egr-1], tis10, tis11 and tis21) and as well as other genes’ expression except gly96 and Stra13 (Fig. 2). This suggests that the expression of these two IEGs by adipogenic stimuli is barely dependent on PKC pathways at least in 3T3-L1 cells. In contrast to PKC pathways, MEK pathway appeared to be more common among the IEGs except for Id-3 and Stra13 genes. Since MAPK and p90 rsk, located at downstream of MEK (MAPKK) phosphorylate and activate Elk-1 and SRF (27, 28), respectively, it is tempting to speculate that PD98059 blocks the expression of IEGs possessing serum response element (SRE) in their promoters. In agreement with this,

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FIG. 2. Analysis of possible signaling pathways responsible for IEGs expression. The effect of MAP kinase, PI3 kinase, and PKC pathways on the IEG expression was assessed. Confluent 3T3-L1 cells were preincubated without or with the indicated inhibitors for one hour prior to the adipogenic challenge. Cells were harvested 30 min later and total RNA was subjected to Northern blot analysis using the DIG-labeled antisense RNA probes corresponding to each gene and resulting bands were visualized with CPD-star (Boehringer Mannheim). Chemiluminescence was quantified by CCD camera (Atto Co., Japan). Relative amount of each RNA is shown using the inhibitor-free RNA as a control. Lanes 1, no inhibitor, lane 2, 10 mM SB203580, lane 3, 50 nM rapamycin, lane 4, 50 mM PD98059, lanes 5, 10 mM Ro-32-0432 and lanes 6, 10 mM LY294002. Data are means from three experiments with standard deviation shown as error bar. IEGs are clustered into three groups according to the susceptibility to different inhibitors (A, B, and C).

SRE(s) is present in the promoters of c-fos (29), of cyr61 (30), and of Thrombospondin-1 (31). However, no SRE sequence has been reported in the promoter region of N-10 (32) and in jun-B (33), though these genes are susceptible to PD98059. This can be interpreted as follows; one possibility is that SRE is present in distant regulatory region of the promoters whose sequence is not yet determined. Another possibility is that MEK

activates unidentified signaling molecules other than SRF or Elk-1, which then leads to the activation of gene expression. Intriguingly, inhibition of PI3 kinase resulted in attenuated expression of gene33 and tis10 genes (Fig. 2B). It is noteworthy that platelet membrane microparticles-dependent tis10 gene expression is also blocked by LY294002 as well as by a PKC inhibitor

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(34). This indicates that there are common pathways in platelet membrane microparticles and in adipogenic hormones signaling which lead to tis10 expression. In contrast to PI3 kinase inhibitor LY294002, rapamycin (which inhibits S6 kinase located at the down stream of PI3 kinase pathways) failed to block the expression of these IEGs (Fig. 2B). This indicates that there may be a signaling molecule(s) other than S6 kinase residing downstream of PI3 kinase that regulates the expression of these IEGs. Future study will be focused on the elucidation of the mechanism by which PI3 kinase controls gene33 and tis10 gene expression. Surprisingly, LY294002 somewhat enhanced Id-3 gene expression (Fig. 2C, lane 6). This implies that PI3 kinase pathways may negatively regulate Id-3 gene, though the real mechanism remains to be addressed. The P38 inhibitor SB203580 did not significantly affect any IEG expression. As p38 pathway is normally activated by stresses such as endotoxin, osmotic shock and metabolic inhibitors but not by mitogen, it is conceivable that the inhibition of the p38 pathway does not affect IEGs expression. In conclusion, our present study demonstrate that DIG-based differential screening is a method suitable for the identification of differentially expressed genes which shows a small difference in the expression level. By combining the subtracted PCR technique (35, 36), it will be very powerful method to detect any differentially expressed genes. Our present data using various kinase inhibitors disclosed a net effect of each signaling molecule on the IEGs expression. The different degree of inhibition observed by a given inhibitor may reflect the difference of the contribution of each kinase pathway to the gene expression. ACKNOWLEDGMENTS We thank Ms. C. Oda for technical assistance. Helix Research Institute is supported by the Ministry of International Trade and Industry (MITI), Chugai Pharmaceutical Co., Fujisawa Pharmaceutical Co., Hitachi Co., Mitsubishi Chemical Co., Nippon Godou Finance Co. (JAFCO), Kyowa Hakko Co., Sumitomo Chemical Co., Taisho Pharmaceutical Co., Yamanouchi Pharmaceutical Co., and Yoshitomi Pharmaceutical Co.

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