Sodium Arsenite Inhibits and Reverses Expression of Adipogenic and Fat Cell-Specific Genes during in Vitro Adipogenesis

Eric M. Wauson, Amy S. Langan and Roseann L. Vorce,1

Center for Environmental Toxicology and Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6260

Received July 14, 2001; accepted September 18, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arsenic causes cancer in humans, but its mechanism of action is unique among known carcinogenic agents. As a naturally occurring component of sediments and ground water, human exposure to arsenic is inevitable, necessitating the establishment of exposure limits. Because cancer is characterized as an imbalance between cell growth and differentiation, it has been hypothesized that arsenic exerts its carcinogenic effect, in part, by perturbing the balance between these antagonistic processes. Previous work in this laboratory has demonstrated that sodium arsenite prevents adipocytic differentiation of C3H 10T1/2 cells, leading to the hypothesis that the underlying mechanism involves downregulation of genes associated with adipogenesis. In support of this hypothesis, it was found that mRNA levels of peroxisome proliferative-activated receptor {gamma} (PPAR{gamma}), CCAAT-enhancer binding protein {alpha} (C/EBP{alpha}), and adipocyte-selective, fatty acid-binding protein (aP2) are decreased in arsenic-treated cells; arsenic-induced phenotypic reversion of differentiated adipocytes correlates with reduced aP2 expression. Arsenic also blocks upregulation of p21Cip1/Waf1, a factor whose expression is tightly regulated during adipogenesis. The differentiating effect of pioglitazone, which induces adipogenesis by activating PPAR{gamma}, is inhibited by arsenic, suggesting that arsenic interferes with adipogenic signaling at or below the level of PPAR{gamma}. Because C/EBP{alpha} is important in the expression of certain keratinocyte-specific genes, the negative effect of arsenic on C/EBP{alpha} might also contribute to the development of skin cancer. PPAR{gamma}, C/EBP{alpha}, and p21Cip1/Waf1 are important in numerous normal and pathological processes, including carcinogenesis, leading us to postulate that perturbation of these factors by arsenic might contribute to the carcinogenic effect of this metalloid.

Key Words: arsenic; differentiation; proliferation; cancer; adipocytes; adipogenesis; aP-2; PPAR{gamma}, C/EBP{alpha}, p21Cip1/Waf1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic ingestion of inorganic arsenic causes basal- and squamous-cell skin carcinomas and also is associated with cancers of the bladder, lung, liver, and kidney (Callen and Headington 1980Go; Hopenhayn-Rich et al., 1998Go; Mazumder et al., 1998Go; Smith et al., 1998Go). In addition to its carcinogenic effects, arsenic exposure has been linked to type II diabetes mellitus (Tseng et al., 2000Go) and cardiovascular diseases (Engel et al., 1994Go), including atherosclerosis, hypertension (Rahman et al., 1999Go), and blackfoot disease (Tseng et al., 1996Go). Since arsenic is a natural constituent of soil and water, some degree of human exposure is inevitable. Therefore, the limits set for the amount of arsenic to which people are exposed must balance safety with the cost incurred by decreasing exposure. Establishing the acceptable concentration of arsenic in the drinking water is an ongoing challenge; this task would be greatly facilitated by discovery of the mechanisms involved in arsenic-induced neoplasia.

Although arsenic exhibits characteristics of both initiators and promoters, its unique spectrum of effects precludes its classification as either (for review, see Kitchin 2001Go). Data from several studies indicate that arsenic alters cell proliferation, and this effect is thought to contribute to its carcinogenicity (Germolec et al., 1997Go, 1998Go; Shimizu et al., 1986Go; Trouba et al., 2000aGo,bGo). Arsenic also inhibits differentiation of cultured cells, including keratinocytes (Kachinskas et al., 1994Go, 1997Go) and adipocytes (Trouba et al., 2000bGo). Since proliferation and differentiation are antagonistic toward one another (Freytag and Geddes, 1992Go), determining the effects of arsenic on the molecular mechanisms underlying each process will contribute to our understanding of the carcinogenicity of arsenic.

Adipogenesis is a well-characterized cellular process, and cultured preadipocyte cell lines have been an invaluable tool in delineating the induction pathway. C/EBPß and C/EBP{delta} are immediate-early targets of adipogenic hormones, and each of these transcription factors is upregulated transiently early in the adipogenic process (Cao et al., 1991Go). In turn, C/EBPß and C/EBP{delta} induce the expression of PPAR{gamma}, a transcription factor absolutely required for adipogenesis (Rosen and Spiegelman 2000Go). A member of the nuclear hormone superfamily, PPAR{gamma} forms a heterodimeric complex with the retinoid-X receptor {alpha} (RXR{alpha}) and induces transcription of a number of fat cell-specific genes, including aP2 and adipsin (Tontonoz et al., 1994aGo).

In addition to physiological stimulation by C/EBPß and C/EBP{delta}, pharmacological induction of PPAR{gamma} can be achieved with dexamethasone (Wu et al., 1996Go). Although endogenous, high affinity PPAR{gamma} ligands have not been identified, a class of antidiabetic agents called thiazolidinediones (TZDs) function as potent PPAR{gamma} ligands (Lehmann et al., 1995Go). C/EBP{alpha}, which is induced by C/EBPß and C/EBP{delta}, is an important transcription factor that works in conjunction with PPAR{gamma} to induce expression of most fat cell-specific genes. In the current model of adipogenesis, PPAR{gamma} induces C/EBP{alpha}; however, C/EBP{alpha} appears to enhance the levels of PPAR{gamma}, thereby setting up a positive feedback loop (reviewed in Rosen et al., 2000). C/EBP{alpha} is sufficient for differentiation of the preadipocyte cell line 3T3-L1 (Freytag and Geddes 1992Go) and appears to be important in maintaining the post-mitotic growth arrest of differentiated adipocytes (Tao and Umek 2000Go).

Expression of the negative growth regulator p21Cip1/Waf1 is regulated tightly during adipogenesis. During the confluent stage prior to induction of differentiation, p21Cip1/Waf1 levels are high, a state consistent with its negative influence on cell proliferation. As cells undergo subsequent clonal expansion, p21Cip1/Waf1 levels decrease, but expression again increases as cells enter post-mitotic growth arrest. The inability of cells to upregulate p21Cip1/Waf1 is postulated to contribute to their continued potential for proliferation and lack of ability to differentiate.

Based on the hypothesis that arsenic inhibits and reverses morphological differentiation of adipocytes by disrupting expression of the panel of genes involved in adipogenesis, including downregulation of fat cell-specific genes, we examined the transcript level of a number of genes associated with adipogenesis and/or growth arrest. Our data support a model in which arsenic perturbs cell programming in a manner consistent with a shift away from differentiation and toward the proliferation pathway.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells and culture conditions.
C3H 10T1/2 cells represent a pluripotent cell line that can be induced to differentiate into adipocytes with the appropriate stimuli. C3H 10T1/2 cells were obtained from the American Type Culture Collection (Rockville, MD) and cultured in DMEM supplemented with 10% FBS, penicillin, and streptomycin (complete media; Life Technologies, Grand Island, NY). The long-term effects of arsenic were assessed by growing cells in the presence or absence of 6 µM sodium arsenite (NaAsO2) for a minimum of 2 months. Cells grown in arsenic for at least 2 months are designated "long-term," and cells grown in the absence of arsenic are designated "control."

Insulin/dexamethasone-induced adipogenesis.
The C3H 10T1/2 cells were subjected to the differentiation protocol as previously described (Trouba et al., 2000bGo), essentially following protocols established by others (Brownell et al., 1996Go; Lu et al., 1992Go). Cells were seeded in DMEM plus 10% FBS. After reaching confluence, the medium was replaced with IDM (DMEM containing 10% FBS, 25 µM dexamethasone (Sigma), and 10 µg/ml insulin (Life Technologies)). After 2 days in IDM, media was then changed to IM (IDM without dexamethasone), and morphological differentiation became evident after another 7–10 days in culture. Cells were photographed using a Nikon Diaphot inverted microscope system (x 25). Unless otherwise stated, analysis was performed at day 12 of the differentiation protocol. In some experiments, mitogenic signaling was inhibited by the addition of the MEK-1 and -2-specific inhibitor U0126 (Favata et al., 1998Go).

Pioglitazone-induced adipogenesis.
Cells were seeded into 48-well plates (15,000 cells/well) in complete media. Once cells reached confluence, fresh DMEM with 10% FBS containing the appropriate concentrations of pioglitazone was added. Fresh pioglitazone supplemented media was added on day 3, and the cultures were incubated for another 4–5 days before adipocyte differentiation was quantified and visualized microscopically.

Assessment of RNA expression.
Following completion of the differentiation protocol, the media was removed, total RNA was isolated using the RNeasy mini-prep system (Qiagen). Following spectrophotometic quantitation, 15- to 20-µg aliquots were fractionated by formaldehyde-agarose gel electrophoresis. Following transfer to nylon membranes, RNA was hybridized to 32P-labeled probes for PPAR{gamma}2 (Tontonoz et al., 1994bGo), C/EBP{alpha} (Christy et al., 1991Go), aP2 (Hunt et al., 1986Go), or p21Waf/Cip1 (Harper et al., 1993Go), all prepared using random primers methodology (Life Technologies). To control for potential variability in sample loading, results were normalized using probes for either {alpha}-tubulin or GAPDH.

Quantification of adipocyte conversion.
Adipocyte differentiation was quantified by staining lipids with Oil Red-O (Sigma) as described previously (Trouba et al., 2000bGo). Briefly, cells were fixed in 10% formalin and stained by immersion in Oil Red-O using the method of Ramirez-Zacarias and coworkers (Ramirez-Zacarias et al., 1992Go). After photography of stained cells, Oil Red-O was extracted and the absorbance at 510 nm was determined spectrophotometrically. Statistical significance was evaluated by analysis of variance using Prism (Graph Pad) followed by Tukey's multiple comparison test (p <.05).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We reported previously that arsenic inhibits morphological differentiation of C3H 10T1/2 cells, but the effect of arsenic on expression of fat-specific genes was not examined (Trouba et al., 2000bGo). To determine if arsenic inhibits adipogenesis at the biochemical level, the mRNA levels of 3 fat cell-specific genes were examined following the dexamethasone/insulin differentiation protocol. As can be seen in Figure 1Go, control C3H 10T1/2 cells induced to differentiate exhibit a high level of aP2, PPAR{gamma} and C/EBP{alpha} (lane 1). In contrast, the mRNA levels of these 3 fat cell-specific genes is greatly diminished in control cells to which 6 µM arsenite has been added (lane 2 for aP2, PPAR{gamma}; lane 3 for C/EBP{alpha}); these cells fail to undergo morphological differentiation (not shown). Similarly, cells treated long term with 6 µM of sodium arsenite and subjected to the differentiation protocol exhibit diminished levels of aP2, PPAR{gamma} and C/EBP{alpha}, regardless of whether arsenic is removed from the media (lane 3 for aP2 and PPAR{gamma}; lane 2 for C/EBP{alpha}) or retained (lane 4). These results indicate that repression of adipogenesis by arsenic involves not only inhibition of lipid accumulation, but also decreased expression of adipocyte marker genes.



View larger version (61K):
[in this window]
[in a new window]
 
FIG. 1. Inhibitory effect of arsenic on the expression of fat cell-specific genes in C3H 10T1/2 cells induced to differentiate. RNA was isolated from cells subjected to the insulin/dexamethasone differentiation protocol and hybridized to radiolabeled probes for aP2, PPAR{gamma}, or C/EBP{alpha}. The data shown represent 1 of 3 experiments performed using either insulin/dexamethasone or pioglitazone to induce differentiation. (A) Results of Northern-blot analysis with each probe. For aP2 and PPAR{gamma}: (lane 1) control cells (morphologically differentiated); (lane 2) cells to which 6 µM As3+ was added at the start of differentiation; (lane 3) cells treated long-term with arsenic but switched to arsenic-free media at the start of differentiation; (lane 4) cells cultured long-term in arsenic and maintained in arsenic-containing media during the differentiation protocol. For C/EBP{alpha} (lanes 1 and 4), identical to aP2 and PPAR{gamma}; (lane 2) cells treated long-term with arsenic but switched to arsenic-free media at the start of differentiation; (lane 3) cells to which 6 µM As3+ was added at the start of differentiation. (B) Results of the Northern blot quantified by densitometry and normalized to the housekeeping genes GAPDH (aP2 and PPAR{gamma}) or tubulin (C/EBP{alpha}).

 
Pioglitazone initiates differentiation by acting directly on PPAR{gamma}, thereby bypassing the requirement for insulin and dexamethasone-activation of C/EBPß and C/EBP{delta}. To determine if arsenic blocks differentiation upstream or downstream of PPAR{gamma}, control C3H 10T1/2 cells or cells treated long term with arsenic were treated with pioglitazone, in either the presence or absence of 6 µM arsenite. As can be seen in Figure 2AGo, pioglitazone-treated control cells assume an adipocyte morphology and accumulate lipid droplets. In contrast, addition of 6 µM arsenite to control cultures completely eliminates the response to pioglitazone. Regardless of whether cells treated long term with arsenic are exposed to pioglitazone in the presence or absence of arsenic, no morphological differentiation is detected. These results are corroborated by Oil Red-O staining of cells treated with 1 to 10 µM pioglitazone (Fig. 2BGo). It can be concluded from these data that signaling events occurring upstream of PPAR{gamma} are not relevant targets of arsenic for its negative effect on adipogenesis.



View larger version (121K):
[in this window]
[in a new window]
 
FIG. 2. Effect of arsenic on C3H 10T1/2 cells induced to differentiate with pioglitizone. Control cells or cells treated long term with 6 µM arsenite were treated with pioglitizone to induce differentiation. (A) Lipid accumulation in cells treated with pioglitizone in the presence or absence of arsenic: Cells were treated as described in the legend to Figure 1Go, except that pioglitizone was used to induce adipocytic differentiation. Lipids were stained with Oil Red-O. (B) Quantification of Oil Red-O accumulation: Oil Red-O was extracted from the cells, quantified by spectrophotometry as described in the text, and depicted as a percentage of the control value (no pioglitazone). Asterisks (*) denote a statistically significant increase in absorbance in control cells compared to arsenic-treated cells.

 
Previous data reported by this laboratory showed that long-term treatment of C3H 10T1/2 cells with arsenic causes an upregulation in MKP-1 (Trouba et al., 2000aGo), which is postulated to increase MAPK activity over time. Since activated MAPK is antagonistic to the process of adipocyte differentiation (Font de Mora et al., 1997Go), we hypothesized that arsenic prevents adipogenesis, in part, by maintaining MAPK in the activated state. To determine if activation of MAPK is required for arsenic to inhibit adipogenesis, the potent and specific MEK-1 and –2 inhibitor U0126 was utilized. As can be seen in Figure 3Go, the inhibition of MEK, and therefore MAPK, does not block the ability of arsenic to inhibit adipogenesis induced by insulin/dexamethasone or pioglitazone, regardless of the concentration of U0126 added. Only control cells respond to the differentiation protocol by accumulating lipids. These results indicate that MAPK activity is not required for inhibition of adipogenesis by arsenic. Interestingly, it appears that U0126 enhances differentiation of control cells, an observation in agreement with the finding that MAPK activation opposes adipogenesis (Font de Mora et al., 1997Go) and with the observation that mitotic clonal expansion is not an essential step in adipogenesis (Qiu et al., 2001Go).



View larger version (71K):
[in this window]
[in a new window]
 
FIG. 3. Effect of MEK inhibition on arsenic-induced inhibition of differentiation. Control cells or cells treated long-term with 6 µM arsenite were induced to differentiate in the presence of various concentrations of U0126. (A) Lipid accumulation was visualized by Oil Red-O staining. (B) Quantification of Oil Red-O accumulation, which was extracted from the cells, quantified by spectrophotometry as described in the text, and depicted as a percentage of the control value (no U0126). The values of control cells are significantly different from those of arsenic-treated cells at all concentrations of U0126, and absorbance at 1, 3, 10, and 30 µM U0126 is significantly higher than that of samples without U0126.

 
Since the addition of sodium arsenite to terminally differentiated adipocytes causes phenotypic reversion characterized by a reduction in accumulated lipids (Trouba et al., 2000bGo), it was predicted that a corresponding decrease in the adipocyte genes aP2, PPAR{gamma} and C/EBP{alpha} would be observed. As shown in Figure 4Go, the mRNAs for all 3 genes are undetectable in control cells and in cells grown long term in 6 µM arsenite (lanes 1 and 2, respectively). Control cells induced to differentiate display an increase in all 3 transcripts at t = 12 days (lane 3), with aP2 levels elevated most noticeably. Further culture of differentiated cells results in a further increase in aP2 and C/EBP{alpha} at day 17 (lane 4). In contrast, aP2 mRNA levels drop dramatically 5 days after addition of arsenic to the differentiated cells (lane 5); levels of PPAR{gamma} and C/EBP{alpha} also decrease, but to a lesser extent. Thus, arsenic-induced phenotypic reversion is accompanied by a decrease in the levels of mRNA corresponding to adipocyte marker genes.



View larger version (41K):
[in this window]
[in a new window]
 
FIG. 4. Reversion effect of arsenic on adipocyte gene expression when added to morphologically differentiated C3H 10T1/2 cells. Cells were treated with insulin/dexamethasone and allowed to differentiate before the addition of 6 µM arsenite on day 12. (A) Northern-blot analysis using probes for aP2, PPAR{gamma}, and C/EBP{alpha}. Lane 1, undifferentiated control cells (day 0); lane 2, undifferentiated cells treated with arsenic long-term (day 0); lane 3, differentiated control cells (day 12); lane 4, differentiated control cells (day 17); lane 5, cells treated at day 12 with 6 µM arsenic (when differentiated) and cultured for 5 additional days before harvest at day 17. (B) Results of Northern blots quantified by densitometry and normalized to GAPDH.

 
In addition to adipocyte-specific genes, the expression of the negative growth regulator p21Cip1/Waf1 was examined. As can be seen in Figure 5Go, mRNA levels of p21Cip1/Waf1 are decreased significantly in cells grown long-term in arsenic as compared to control cells under conditions of confluence. The inability of arsenic-treated cells to increase p21Cip1/Waf1 expression during density-dependent growth arrest is consistent with a decreased capacity to undergo terminal differentiation. The effects of arsenic on adipogenic and fat cell-specific gene expression are summarized in Figure 6Go.



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 5. Effect of arsenic on p21Cip1/Waf1 mRNA levels in C3H 10T1/2 cells. RNA was isolated from confluent cells (control cells or cells treated long-term with 6 µM arsenic) and subjected to Northern-blot analysis, using a probe for p21Cip1/Waf1. (A) The results from 2 separate samples for control and arsenic-treated cells are shown. (B) Densitometric quantitation and normalization of p21Cip1/Waf1 mRNA to {alpha}-tubulin.

 


View larger version (31K):
[in this window]
[in a new window]
 
FIG. 6. Influence of arsenic on adipogenic signal transduction. As denoted by the bold downward arrows, arsenic treatment results in decreased expression of the key adipogenic factors PPAR{gamma} and C/EBP{alpha} and the fat cell-specific gene aP2.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have shown previously that arsenic inhibits and reverses morphological differentiation of C3H 10T1/2 pre-adipocytes as assessed by lipid accumulation (Trouba et al., 2000bGo). However, these studies did not distinguish between an effect of arsenic to decrease lipids secondary to effects on adipocyte biochemistry (e.g., inhibition of lipogenesis or glucose uptake) versus an arsenic-dependent alteration in adipogenic cellular programming. The results described herein indicate clearly that arsenic decreases expression of fat cell-specific and adipogenic genes, thereby supporting the hypothesis that arsenic perturbs regulatory processes fundamental to maintaining the appropriate balance between proliferation and differentiation.

The finding that arsenic inhibits PPAR{gamma} expression could be an important key in determining the mechanism by which this metalloid perturbs normal control of growth and differentiation. PPAR{gamma} can be considered a "master switch" in adipogenesis, acting to coordinate expression of numerous genes involved in the differentiation process. In addition, PPAR{gamma} initiates withdrawal from the cell cycle, an event necessary in the induction of adipogenesis (Altiok et al., 1997Go). In smooth muscle cells, PPAR{gamma} activation results in accumulation of cells in G0/G1, an effect accompanied by upregulation of the negative growth regulator p27Kip1 (Wakino et al., 2000Go). It follows that arsenic-treated cells exhibiting decreased PPAR{gamma} expression are predicted to be less likely to enter G0/G1 and less likely to exhibit a normal increase in p27Kip1 expression when induced to differentiate. Therefore, inhibited PPAR{gamma} expression is a reasonable explanation for our previous data showing that arsenic treated cells are primed to enter the cell cycle and display decreased p27Kip1 levels (Trouba et al., 2000aGo,bGo).

Our finding that arsenic causes decreased expression of PPAR{gamma} also is of potential importance in understanding the mechanisms underlying diverse pathologies induced by arsenic. In exposed human populations, arsenic exposure has been linked to type II diabetes mellitus (Rahman et al., 1998Go; Tseng et al., 2000Go) and hypertension (Rahman et al., 1999Go), and downregulation of PPAR{gamma} has been correlated with both of these disease states (Barroso et al., 1999Go). Conversely, PPAR{gamma} agonists inhibit the growth and/or initiate differentiation of a variety of cultured tumor cell lines, including colorectal, breast, and prostate cancer cells (Brockman et al., 1998Go; Elstner et al., 1998Go; Kubota et al., 1998Go; Mueller et al., 1998Go). Therefore, it is intriguing to speculate that many of the carcinogenic and noncarcinogenic pathologies resulting from arsenic exposure might result from downregulation of PPAR{gamma}.

Another piece of evidence indicating that PPAR{gamma} is an important target of arsenic is provided by the results of experiments using pioglitazone to induce adipogenesis. Direct activation of PPAR{gamma} circumvents the requirement for hormonally induced expression of C/EBPß and C/EBP{alpha}, two factors that contribute to PPAR{gamma} induction. Because arsenic efficaciously inhibits adipogenesis induced by this PPAR{gamma} agonist, it can be concluded that upstream signaling events are not required for the inhibitory effects of arsenic on differentiation.

In cells induced to undergo adipogenesis, treatment with arsenic tips the balance away from differentiation and toward proliferation. At the molecular level, adipogenesis is inhibited by phosphorylation of PPAR{gamma} by MAP kinase (Hu et al., 1996Go), and arsenic potentially increases MAPK activity secondary to down regulated MKP-1 expression (Trouba et al., 2000aGo). Therefore, it is conceivable that arsenic blocks adipogenesis simply by maintaining cells in a state of proliferative activity. This leads to a major question of whether arsenic inhibits adipogenesis indirectly by its ability to maintain cells in a mitogenically competent state, or by a more direct mechanism involving adipogenic processes. We found that UO126-mediated inhibition of mitogenic signaling does not abrogate the differentiation inhibition effect of arsenic, thereby supporting the hypothesis that arsenic interferes with the molecular events leading to differentiation.

For full phenotypic differentiation of adipocytes, expression of both PPAR{gamma} and C/EBP{alpha} is required (El-Jack et al., 1999Go). In addition to playing a pivotal role in establishment of insulin-sensitive glucose transport, C/EBP{alpha} is necessary for maintenance of post-mitotic growth arrest in adipocytes (Tao and Umek 2000Go; Timchenko et al., 1996Go). Downregulation of C/EBP{alpha}, accomplished by expression of antisense RNA, enables quiescent cells to reenter the cell cycle (Tao and Umek 2000Go), thereby illustrating the importance of C/EBP{alpha} in maintaining proliferative inactivity. Because C/EBP{alpha} positively regulates protein levels of p21Waf1/Cip1, (Timchenko et al., 1996Go), the abnormally low level of p21Waf1/Cip1 seen in arsenic-treated cells (Fig 5Go) might be a consequence of arsenic-induced down regulation of C/EBP{alpha}. Using animals genetically engineered to lack C/EBP{alpha}, a correlation has been made between loss of C/EBP{alpha}, decreased p21Waf1/Cip1 levels, and unregulated cell proliferation in vivo (Timchenko et al., 1997Go).

There is increasing evidence that C/EBPs, including C/EBP{alpha}, are important in regulating epidermal differentiation (Maytin and Habener 1998Go; Maytin et al., 1999Go; Oh and Smart 1998Go; Swart et al., 1997Go; Zhu et al., 1999Go). C/EBP{alpha} and C/EBPß are greatly reduced in squamous cell carcinomas when compared to control cells (Oh and Smart 1998Go), establishing a link between changes in C/EBP expression and malignant transformation. In light of the fact that arsenic causes primarily skin cancer, arsenic-induced alterations in C/EBP{alpha} could contribute to inhibition of differentiation.

Like other members of the family of cyclin-dependent kinase inhibitors, p21Cip1/Waf1 plays an important role in regulating cell cycle progression by inhibiting the cyclin-dependent kinases (CDKs). A putative target gene of C/EBP{alpha}, p21Cip1/Waf1 is upregulated following induction of C/EBP{alpha} (Heath et al., 2000Go; Timchenko et al., 1996Go). Furthermore, the proliferative inhibition exerted by C/EBP{alpha} is mediated through p21Cip1/Waf1 (Timchenko et al., 1996Go). Therefore, decreased p21Cip1/Waf1 expression in arsenic-treated cells might be a consequence of downregulated C/EBP{alpha} expression. Regardless of the mechanism, the changes observed in cellular p21Cip1/Waf1 expression following arsenic exposure are consistent with maintenance of proliferative competence during conditions favorable for differentiation. The loss of p21Cip1/Waf1-dependent negative regulation has been implicated in the cocarcinogenic property of arsenic (Vogt and Rossman 2001Go).

The results of the present study corroborate our earlier report that arsenic inhibits morphological differentiation of C3H 10T1/2 cells into adipocytes. The data extend our understanding of this effect by demonstrating that arsenic disrupts adipogenesis via perturbations of the cellular programming underlying this phenomenon. It is anticipated that this information will contribute to a definitive identification of the molecular target(s) of arsenic that lead to its adverse effects.


    ACKNOWLEDGMENTS
 
The authors thank Dr. Robert E. Lewis for the adipocyte cDNAs and the pioglitazone and Lisa Crandall for thoughtful critique of the manuscript. This work was supported by National Institutes of Health grant #ES07505.


    NOTES
 
1 To whom correspondence should be addressed at Pfizer Global Research and Development, 2200 Commonwealth Drive, Ann Arbor, MI 48105. Fax: (734) 622-3300. E-mail: roseann.vorce{at}pfizer.com. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Altiok, S., Xu, M., and Spiegelman, B. (1997). PPAR{gamma} induces cell cycle withdrawal: Inhibition of E2F/DP DNA-binding activity via downregulation of PP2A. Genes Dev. 11, 1987–1998.[Abstract/Free Full Text]

Barroso, I., Gurnell, M., Crowley, V., Agostini, M., Schwabe, J., Soos, M., Maslen, G., Williams, T., Lewis, H., Schafer, A., Chatterjee, V., and O'Rahilly, S. (1999). Dominant negative mutations in human PPAR{gamma} associated with severe insulin resistance, diabetes mellitus, and hypertension. Nature 402, 880–883.[ISI][Medline]

Brockman, J., Gupta, R., and Dubois, R. (1998). Activation of PPAR{gamma} leads to inhibition of anchorage-independent growth of human colorectal cancer cells. Gastroenterology 115, 1049–1055.[ISI][Medline]

Brownell, H. L., Narsimhan, R. P., Corbley, M. J., Mann, V. M., Whitfield, J. F., and Raptis, L. (1996). Ras is involved in gap-junction closure in proliferating fibroblasts or preadipocytes but not in differentiated adipocytes. DNA Cell Biol. 15, 443–451.[ISI][Medline]

Callen, J. P., and Headington, J. (1980). Bowen's and non-Bowen's squamous intraepidermal neoplasia of the skin. Relationship to internal malignancy. Arch. Dermatol. 116, 422–426.[Abstract]

Cao, Z., Umek, R., and McKnight, S. (1991). Regulated expression of three C/EBP isoforms during adipose conversion of 3T3–L1 cells. Genes Dev. 5, 1538–1552.[Abstract]

Christy, R., Kaestner, K., Geiman, D., and Lane, M. (1991). CCAAT/enhancer binding protein gene promoter: Binding of nuclear factors during differentiation of 3T3–L1 preadipocytes. Proc. Natl. Acad. Sci. U.S.A. 88, 2593–2597.[Abstract]

El-Jack, A., Hamm, J., Pilch, P., and Farmer, S. (1999). Reconstitution of insulin-sensitive glucose transport in fibroblasts requires expression of both PPAR{gamma} and C/EBP{alpha}. J. Biol. Chem. 274, 7946–7951.[Abstract/Free Full Text]

Elstner, E., Muller, C., Koshizuka, K., Williamson, E., Park, D., Asou, H., Shintaku, P., Said, J., Heber, D., and Koeffler, H. (1998). Ligands for peroxisome proliferator-activated receptor-{gamma} and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. Proc. Natl. Acad. Sci. U.S.A. 95, 8806–8811.[Abstract/Free Full Text]

Engel, R., Hopenhayn-Rich, C., Receveur, O., and Smith, A. (1994). Vascular effects of chronic arsenic exposure: A review. Epidemiol. Rev. 16, 184–209.[ISI][Medline]

Favata, M., Horiuchi, K., Manos, E., Daulerio, A., Stradley, D., Feeser, W., Van Dyk, D., Pitts, W., Earl, R., Hobbs, F., Copeland, R., Magolda, R., Scherle, P., and Trzaskos, J. (1998). Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273, 18623–18632.[Abstract/Free Full Text]

Font de Mora, J., Porras, A., Ahn, N., and Santos, E. (1997). Mitogen-activated protein kinase activation is not necessary for, but antagonizes, 3T3–L1 adipocytic differentiation. Mol. Cell. Biol. 17, 6068–6075.[Abstract]

Freytag, S. O., and Geddes, T. J. (1992). Reciprocal regulation of adipogenesis by Myc and C/EBP{alpha}. Science 256, 379–382.[ISI][Medline]

Germolec, D. R., Spalding, J., Boorman, G. A., Wilmer, J. L., Yoshida, T., Simeonov, P. P., Bruccoleri, A., Kayama, F., Gaido, K., Tennant, R., Burleson, F., Dong, W., Lang, R. W., and Luster, M. I. (1997). Arsenic can mediate skin neoplasia by chronic stimulation of keratinocyte-derived growth factors. Mutat. Res. 386, 209–218.[ISI][Medline]

Germolec, D. R., Spalding, J., Yu, H. S., Chen, G. S., Simeonova, P. P., Humble, M. C., Bruccoleri, A., Boorman, G. A., Foley, J. F., Yoshida, T., and Luster, M. I. (1998). Arsenic enhancement of skin neoplasia by chronic stimulation of growth factors. Am. J. Pathol. 153, 1775–1785.[Abstract/Free Full Text]

Harper, J., Adami, G., Wei, N., Keyomarsi, K., and Elledge, S. (1993). The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell Growth Differ. 75, 805–816.

Heath, V., Gillespie, D., and Crouch, D. (2000). Inhibition of adipocyte differentiation by cMyc is not accompanied by alterations in cell-cycle control. Biochem. Biophys. Res. Commun. 269, 438–443.[ISI][Medline]

Hopenhayn-Rich, C., Biggs, M. L., and Smith, A. H. (1998). Lung and kidney cancer mortality associated with arsenic in drinking water in Cordoba, Argentina. Int. J. Epidemiol. 27, 561–569.[Abstract]

Hu, E., Kim, J., Sarraf, P., and Spiegelman, B. (1996). Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPAR{gamma}. Science 274, 2100–2103.[Abstract/Free Full Text]

Hunt, C., Ro, J., Dobson, D., Min, H., and Spiegelman, B. (1986). Adipocyte P2 gene: Developmental expression and homology of 5`-flanking sequences among fat cell-specific genes. Proc. Natl. Acad. Sci. U.S.A. 83, 3786–3790.[Abstract]

Kachinskas, D. J., Phillips, M. A., Qin, Q., Stokes, J. D., and Rice, R. H. (1994). Arsenate perturbation of human keratinocyte differentiation. Cell Growth Differ. 5, 1235–1241.[Abstract]

Kachinskas, D. J., Qin, Q., Phillips, M. A., and Rice, R. H. (1997). Arsenate suppression of human keratinocyte programming. Mutat. Res. 386, 253–261.[ISI][Medline]

Kitchin, K. (2001). Recent advances in arsenic carcinogenesis: Modes of action, animal model systems, and methylated arsenic metabolites. Toxicol. Appl. Pharmacol. 172, 249–261.[ISI][Medline]

Kubota, T., Koshizuka, K., Williamson, E., Asou, H., Said, J., Holden, S., Miyoshi, I., and Koeffler, H. (1998). Ligand for peroxisome proliferator-activated receptor {gamma} (troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo. Cancer Res. 58, 3344–3352.[Abstract]

Lehmann, J., Moore, L., Smith-Oliver, T., Wilkison, W., Willson, T., and Kliewer, S. (1995). An antidiabetic thiazolidinedione is a high-affinity ligand for peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). J. Biol. Chem. 270, 12953–12956.[Abstract/Free Full Text]

Lu, Y., Raptis, L., Anderson, S., Corbley, M. J., Zhou, Y. C., Pross, H., and Haliotis, T. (1992). Ras modulates commitment and maturation of 10T1/2 fibroblasts to adipocytes. Biochem. Cell. Biol. 70, 1249–1257.[ISI][Medline]

Maytin, E., and Habener, J. (1998). Transcription factors C/EBP{alpha}, C/EBPß, and CHOP (Gadd153) expressed during the differentiation program of keratinocytes in vitro and in vivo. J. Invest. Dermatol. 110, 238–246.[Abstract]

Maytin, E., Lin, J., Krishnamurthy, R., Batchvarova, N., Ron, D., Mitchell, P., and Habener, J. (1999). Keratin 10 gene expression during differentiation of mouse epidermis requires transcription factors C/EBP and AP-2. Dev. Biol. 216, 164–181.[ISI][Medline]

Mazumder, D. N., Das Gupta, J., Santra, A., Pal, A., Ghose, A., and Sarkar, S. (1998). Chronic arsenic toxicity in west Bengal—the worst calamity in the world. J. Indian Med. Assoc. 96, 4–7, 18.[Medline]

Mueller, E., Sarraf, P., Tontonoz, P., Evans, R., Martin, K., Zhang, M., Fletcher, C., Singer, S., and Spiegelman, B. (1998). Terminal differentiation of human breast cancer through PPAR{gamma}. Mol. Cell. 1, 465–470.[ISI][Medline]

Oh, H., and Smart, R. (1998). Expression of CCAAT/enhancer binding proteins (C/EBP) is associated with squamous differentiation in epidermis and isolated primary keratinocytes and is altered in skin neoplasms. J. Invest. Dermatol. 110, 939–945.[Abstract]

Qiu, Z., Wei, Y., Chen, N., Jiang, M., Wu, J., and Liao, K. (2001). DNA synthesis and mitotic clonal expansion is not a required step for 3T3–L1 preadipocyte differentiation into adipocytes. J. Biol. Chem. 276, 11988–11995.[Abstract/Free Full Text]

Rahman, M., Tondel, M., Ahmad, S., and Axelson, O. (1998). Diabetes mellitus associated with arsenic exposure in Bangladesh. Am. J. Epidemiol. 148, 198–203.[Abstract]

Rahman, M., Tondel, M., Ahmad, S., Chowdhury, I., Faruquee, M., and Axelson, O. (1999). Hypertension and arsenic exposure in Bangladesh. Hypertension 33, 74–78.[Abstract/Free Full Text]

Ramirez-Zacarias, J. L., Castro-Munozledo, F., and Kuri-Harcuch, W. (1992). Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry 97, 493–497.[ISI][Medline]

Rosen, E., and Spiegelman, B. (2000). Molecular regulation of adipogenesis. Annu. Rev. Cell. Dev. Biol. 16, 145–171.[ISI][Medline]

Shimizu, M., Torti, F., and Roth, R. (1986). Characterization of the insulin and insulin-like growth factor receptors and responsitivity of a fibroblast/adipocyte cell line before and after differentiation. Biochem. Biophys. Res. Commun. 137, 552–558.[ISI][Medline]

Smith, A. H., Goycolea, M., Haque, R., and Biggs, M. L. (1998). Marked increase in bladder and lung cancer mortality in a region of northern Chile, due to arsenic in drinking water. Am. J. Epidemiol. 147, 660–669.[Abstract]

Swart, G., van Groningen, J., van Ruissen, F., Bergers, M., and Schalkwijk, J. (1997). Transcription factor C/EBP{alpha}: Novel sites of expression and cloning of the human gene. Biol .Chem. 378, 373–379.[ISI][Medline]

Tao, H., and Umek, R. (2000). C/EBP{alpha} is required to maintain post-mitotic growth arrest in adipocytes. DNA Cell Biol. 19, 9–18.[ISI][Medline]

Timchenko, N., Harris, T., Wilde, M., Bilyeu, T., Burgess-Beusse, B., Finegold, M., and Darlington, G. (1997). CCAAT/enhancer binding protein {alpha} regulates p21 protein and hepatocyte proliferation in newborn mice. Mol. Cell Biol. 17, 7353–7361.[Abstract]

Timchenko, N., Wilde, M., Nakanishi, M., Smith, J., and Darlington, G. (1996). CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}) inhibits cell proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein. Genes Dev. 10, 804–815.[Abstract]

Tontonoz, P., Graves, R., Budavari, A., Erdjument-Bromage, H., Lui, M., Hu, E., Tempst, P., and Spiegelman, B. (1994a). Adipocyte-specific transcription factor ARF6 is a heterodimeric complex of two nuclear hormone receptors, PPAR{gamma} and RXR{alpha}. Nucleic Acids Res. 22, 5628–5634.[Abstract]

Tontonoz, P., Hu, E., Graves, R., Budavari, A., and Spiegelman, B. (1994b). mPPAR {gamma} 2: Tissue-specific regulator of an adipocyte enhancer. Genes Dev. 8, 1224–1234.[Abstract]

Trouba, K. J., Wauson, E. M., and Vorce, R. L. (2000a). Sodium arsenite-induced dysregulation of proteins involved in proliferative signaling. Toxicol. Appl. Pharmacol. 164, 161–170.[ISI][Medline]

Trouba, K. J., Wauson, E. M., and Vorce, R. L. (2000b). Sodium arsenite inhibits terminal differentiation of murine C3H 10T1/2 preadipocytes. Toxicol. Appl. Pharmacol. 168, 25–35.[ISI][Medline]

Tseng, C., Chong, C., Chen, C., and Tai, T. (1996). Dose-response relationship between peripheral vascular disease and ingested inorganic arsenic among residents in blackfoot disease-endemic villages in Taiwan. Atherosclerosis 120, 125–133.[ISI][Medline]

Tseng, C. H, Tai, T., Chong, C., Tseng, C. P., Lai, M., Lin, B., Chiou, H., Hsueh, Y., Hsu, K., and Chen, C. (2000). Long-term arsenic exposure and incidence of non-insulin-dependent diabetes mellitus: A cohort study in arseniasis-hyperendemic villages in Taiwan. Environ. Health Perspect. 108, 847–851.[ISI][Medline]

Vogt, B., and Rossman, T. (2001). Effects of arsenite on p53, p21, and cyclin-D expression in normal human fibroblasts—a possible mechanism for arsenite's co-mutagenicity. Mutat. Res. 478, 159–168.[ISI][Medline]

Wakino, S., Kintscher, U., Kim, S., Yin, F., Hsueh, W., and Law, R. (2000). Peroxisome proliferator-activated receptor {gamma} ligands inhibit retinoblastoma phosphorylation and G1–> S transition in vascular smooth muscle cells. J. Biol. Chem. 275, 22435–22441.[Abstract/Free Full Text]

Wu, Z., Buchner, N. L., and Farmer, S. R. (1996). Induction of peroxisome proliferator-activated receptor gamma during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBP beta, C/EBP delta, and glucocorticoids. Mol. Cell. Biol. 16, 4128–4136.[Abstract]

Zhu, S., Oh, H., Shim, M., Sterneck, E., Johnson, P., and Smart, R. (1999). C/EBPbeta modulates the early events of keratinocyte differentiation involving growth arrest and keratin 1 and keratin 10 expression. Mol. Cell. Biol. 19, 7181–7190.[Abstract/Free Full Text]