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
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ABSTRACT |
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Key Words: arsenic; differentiation; proliferation; cancer; adipocytes; adipogenesis; aP-2; PPAR, C/EBP
, p21Cip1/Waf1.
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INTRODUCTION |
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Although arsenic exhibits characteristics of both initiators and promoters, its unique spectrum of effects precludes its classification as either (for review, see Kitchin 2001). Data from several studies indicate that arsenic alters cell proliferation, and this effect is thought to contribute to its carcinogenicity (Germolec et al., 1997
, 1998
; Shimizu et al., 1986
; Trouba et al., 2000a
,b
). Arsenic also inhibits differentiation of cultured cells, including keratinocytes (Kachinskas et al., 1994
, 1997
) and adipocytes (Trouba et al., 2000b
). Since proliferation and differentiation are antagonistic toward one another (Freytag and Geddes, 1992
), 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 are immediate-early targets of adipogenic hormones, and each of these transcription factors is upregulated transiently early in the adipogenic process (Cao et al., 1991
). In turn, C/EBPß and C/EBP
induce the expression of PPAR
, a transcription factor absolutely required for adipogenesis (Rosen and Spiegelman 2000
). A member of the nuclear hormone superfamily, PPAR
forms a heterodimeric complex with the retinoid-X receptor
(RXR
) and induces transcription of a number of fat cell-specific genes, including aP2 and adipsin (Tontonoz et al., 1994a
).
In addition to physiological stimulation by C/EBPß and C/EBP, pharmacological induction of PPAR
can be achieved with dexamethasone (Wu et al., 1996
). Although endogenous, high affinity PPAR
ligands have not been identified, a class of antidiabetic agents called thiazolidinediones (TZDs) function as potent PPAR
ligands (Lehmann et al., 1995
). C/EBP
, which is induced by C/EBPß and C/EBP
, is an important transcription factor that works in conjunction with PPAR
to induce expression of most fat cell-specific genes. In the current model of adipogenesis, PPAR
induces C/EBP
; however, C/EBP
appears to enhance the levels of PPAR
, thereby setting up a positive feedback loop (reviewed in Rosen et al., 2000). C/EBP
is sufficient for differentiation of the preadipocyte cell line 3T3-L1 (Freytag and Geddes 1992
) and appears to be important in maintaining the post-mitotic growth arrest of differentiated adipocytes (Tao and Umek 2000
).
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.
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MATERIALS AND METHODS |
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Insulin/dexamethasone-induced adipogenesis.
The C3H 10T1/2 cells were subjected to the differentiation protocol as previously described (Trouba et al., 2000b), essentially following protocols established by others (Brownell et al., 1996
; Lu et al., 1992
). 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 710 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., 1998
).
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 45 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 PPAR2 (Tontonoz et al., 1994b
), C/EBP
(Christy et al., 1991
), aP2 (Hunt et al., 1986
), or p21Waf/Cip1 (Harper et al., 1993
), all prepared using random primers methodology (Life Technologies). To control for potential variability in sample loading, results were normalized using probes for either
-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., 2000b). 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., 1992
). 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).
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RESULTS |
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DISCUSSION |
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The finding that arsenic inhibits PPAR expression could be an important key in determining the mechanism by which this metalloid perturbs normal control of growth and differentiation. PPAR
can be considered a "master switch" in adipogenesis, acting to coordinate expression of numerous genes involved in the differentiation process. In addition, PPAR
initiates withdrawal from the cell cycle, an event necessary in the induction of adipogenesis (Altiok et al., 1997
). In smooth muscle cells, PPAR
activation results in accumulation of cells in G0/G1, an effect accompanied by upregulation of the negative growth regulator p27Kip1 (Wakino et al., 2000
). It follows that arsenic-treated cells exhibiting decreased PPAR
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
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., 2000a
,b
).
Our finding that arsenic causes decreased expression of PPAR 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., 1998
; Tseng et al., 2000
) and hypertension (Rahman et al., 1999
), and downregulation of PPAR
has been correlated with both of these disease states (Barroso et al., 1999
). Conversely, PPAR
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., 1998
; Elstner et al., 1998
; Kubota et al., 1998
; Mueller et al., 1998
). Therefore, it is intriguing to speculate that many of the carcinogenic and noncarcinogenic pathologies resulting from arsenic exposure might result from downregulation of PPAR
.
Another piece of evidence indicating that PPAR is an important target of arsenic is provided by the results of experiments using pioglitazone to induce adipogenesis. Direct activation of PPAR
circumvents the requirement for hormonally induced expression of C/EBPß and C/EBP
, two factors that contribute to PPAR
induction. Because arsenic efficaciously inhibits adipogenesis induced by this PPAR
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 by MAP kinase (Hu et al., 1996
), and arsenic potentially increases MAPK activity secondary to down regulated MKP-1 expression (Trouba et al., 2000a
). 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 and C/EBP
is required (El-Jack et al., 1999
). In addition to playing a pivotal role in establishment of insulin-sensitive glucose transport, C/EBP
is necessary for maintenance of post-mitotic growth arrest in adipocytes (Tao and Umek 2000
; Timchenko et al., 1996
). Downregulation of C/EBP
, accomplished by expression of antisense RNA, enables quiescent cells to reenter the cell cycle (Tao and Umek 2000
), thereby illustrating the importance of C/EBP
in maintaining proliferative inactivity. Because C/EBP
positively regulates protein levels of p21Waf1/Cip1, (Timchenko et al., 1996
), the abnormally low level of p21Waf1/Cip1 seen in arsenic-treated cells (Fig 5
) might be a consequence of arsenic-induced down regulation of C/EBP
. Using animals genetically engineered to lack C/EBP
, a correlation has been made between loss of C/EBP
, decreased p21Waf1/Cip1 levels, and unregulated cell proliferation in vivo (Timchenko et al., 1997
).
There is increasing evidence that C/EBPs, including C/EBP, are important in regulating epidermal differentiation (Maytin and Habener 1998
; Maytin et al., 1999
; Oh and Smart 1998
; Swart et al., 1997
; Zhu et al., 1999
). C/EBP
and C/EBPß are greatly reduced in squamous cell carcinomas when compared to control cells (Oh and Smart 1998
), 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
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, p21Cip1/Waf1 is upregulated following induction of C/EBP
(Heath et al., 2000
; Timchenko et al., 1996
). Furthermore, the proliferative inhibition exerted by C/EBP
is mediated through p21Cip1/Waf1 (Timchenko et al., 1996
). Therefore, decreased p21Cip1/Waf1 expression in arsenic-treated cells might be a consequence of downregulated C/EBP
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 2001
).
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.
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ACKNOWLEDGMENTS |
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NOTES |
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