Regulation of basal autophagy by calmodulin availability.

Macroautophagy (hereafter autophagy) is a process that degrades cellular components to maintain homeostasis. The Ca2+ sensor calmodulin (CaM) regulates numerous cell functions but is a limiting factor due to its insufficient availability for all target proteins. However, evidence that CaM availability regulates basal autophagy is lacking. Here, we have tested this hypothesis. CaM antagonists W-7, trifluoperazine and CGS9343b cause autophagosome accumulation and inhibit basal autophagic flux in the same manner as does chloroquine. These reagents promote the activity of AMP-activated protein kinase (AMPK) but not that of the mechanistic target of rapamycin (mTOR). Competitive binding assays using CaM sensors with different Ca2+ dependencies showed that chloroquine directly binds CaM in a Ca2+ -dependent fashion. The CaM antagonists have disparate effects on cytoplasmic Ca2+ , triggering from none to robust signals, indicating that their consistent inhibition of autophagy is due to inhibition of CaM and not Ca2+ . Chelating intracellular Ca2+ reduces the effect of the CaM antagonists to accumulate LC3-II, indicating that they do so by inhibiting CaM-dependent activities at basal Ca2+ level. The CaM antagonists cause lysosomal alkalinisation. Consistently, buffering CaM with a high-affinity CaM-binding protein that binds CaM at resting Ca2+ level increases lysosomal pH. Enhanced CaM buffering using a chimeric protein that contains two high-affinity CaM-binding sites that can collectively bind CaM at a large range of Ca2+ further increases lysosomal pH and increases LC3-II accumulation and AMPK activity, but not that of mTOR. These data demonstrate that CaM availability is required for basal autophagy.

A novel assay to assess the effects of estrogen on the cardiac calmodulin binding equilibrium.

AIMS: The Ca2+-binding protein calmodulin (CaM) modulates numerous target proteins but is produced insufficiently to bind all of them, generating a limiting CaM equilibrium. Menopause increases cardiac morbidity; however, it is unknown if the cardiac CaM equilibrium is affected by estrogen. We devised an assay to assess the effects of ovariectomy and estrogen treatment on the cardiac CaM equilibrium. MATERIALS AND METHODS: Sprague-Dawley rats received sham surgery or ovariectomy, followed by 2-week treatment with vehicle or 17ß-estradiol. Ca2+-saturated left ventricular (LV) lysates were processed through CaM sepharose columns, which retained CaM-binding proteins unoccupied by endogenous CaM. Eluants therefrom were subjected to a competitive binding assay against purified CaM and a CaM biosensor to assess the amounts of unoccupied CaM-binding sites. LV cellular composition was assessed by immunohistochemistry. KEY FINDINGS: LV eluants processed from sham animals reduce biosensor response by ~32%, indicating baseline presence of unoccupied CaM-binding sites and a limiting CaM equilibrium. Ovariectomy exacerbates the limiting CaM equilibrium, reducing biosensor response by ~65%. 17ß-estradiol treatment equalizes the difference between sham and ovariectomized animals. These changes reflect whole tissue responses and are not mirrored by changes in total surface areas of cardiomyocytes and fibroblasts. Consistently, Ca2+-dependent, but not Ca2+-independent, interaction between CaM and the cardiac inositol trisphosphate receptor (IP3R) is reduced following ovariectomy and is restored by subsequent 17ß-estradiol treatment. SIGNIFICANCE: Our assay provides a new parameter to assess tissue CaM equilibrium. The exacerbated limiting CaM equilibrium following estrogen loss may contribute to cardiac morbidity and is prevented by estrogen treatment.

Hippocampal mitogen-activated protein kinase phosphatase-1 regulates behavioral and systemic effects of chronic corticosterone administration.

Clinical reports indicate a bidirectional relationship between mental illness and chronic systemic diseases. However, brain mechanisms linking chronic stress and development of mood disorders to accompanying peripheral organ dysfunction are still not well characterized in animal models. In the current study, we investigated whether activation of hippocampal mitogen-activated protein kinase phosphatase-1 (MKP-1), a key factor in depression pathophysiology, also acts as a mediator of systemic effects of stress. First, we demonstrated that treatment with the glucocorticoid receptor (GR) agonist dexamethasone or acute restraint stress (ARS) significantly increased Mkp-1 mRNA levels within the rat hippocampus. Conversely, administration of the GR antagonist mifepristone 30 min before ARS produced a partial blockade of Mkp-1 upregulation, suggesting that stress activates MKP-1, at least in part, through upstream GR signaling. Chronic corticosterone (CORT) administration evoked comparable increases in hippocampal MKP-1 protein levels and produced a robust increase in behavioral emotionality. In addition to behavioral deficits, chronic CORT treatment also produced systemic pathophysiological effects. Elevated levels of renal inflammation protein markers (NGAL and IL18) were observed suggesting tissue damage and early kidney impairment. In a rescue experiment, the effects of CORT on development of depressive-like behaviors and increased NGAL and IL18 protein levels in the kidney were blocked by CRISPR-mediated knockdown of hippocampal Mkp-1 prior to CORT exposure. In sum, these findings further demonstrate that MKP-1 is necessary for development of enhanced behavioral emotionality, while also suggesting a role in stress mechanisms linking brain dysfunction and systemic illness such as kidney disease.

Corticosterone as a Potential Confounding Factor in Delineating Mechanisms Underlying Ketamine's Rapid Antidepressant Actions.

Recent research into the rapid antidepressant effect of subanesthetic doses of ketamine have identified a series of relevant protein cascades activated within hours of administration. Prior to, or concurrent with, these activation cascades, ketamine treatment generates dissociative and psychotomimetic side effects along with an increase in circulating glucocorticoids. In rats, we observed an over 3-fold increase in corticosterone levels in both serum and brain tissue, within an hour of administration of low dose ketamine (10 mg/kg), but not with (2R, 6R)-hydroxynorketamine (HNK) (10 mg/kg), a ketamine metabolite shown to produce antidepressant-like action in rodents without inducing immediate side-effects. Hippocampal tissue from ketamine, but not HNK, injected animals displayed a significant increase in the expression of sgk1, a downstream effector of glucocorticoid receptor signaling. To examine the role conscious sensation of ketamine's side effects plays in the release of corticosterone, we assessed serum corticosterone levels after ketamine administration while under isoflurane anesthesia. Under anesthesia, ketamine failed to increase circulating corticosterone levels relative to saline controls. Concurrent with its antidepressant effects, ketamine generates a release of glucocorticoids potentially linked to disturbing cognitive side effects and the activation of distinct molecular pathways which should be considered when attempting to delineate the molecular mechanisms of its antidepressant function.

Loss of the nutrient receptor Tas1R3 reduces atherosclerotic plaque accumulation and hepatic steatosis in ApoE-/- mice.

The taste receptor type I (Tas1R) family consists of three G protein-coupled receptors (T1R1, T1R2, and T1R3) that form heterodimers recognizing sweet compounds (T1R2/T1R3) or amino acids (T1R1/T1R3). These receptors are nutrient sensors that facilitate appropriate physiological responses with nutrient availability. However, their contribution to the development of pathologies associated with overnutrition (e.g., atherosclerosis) is unclear. The aim of the present study was to determine if T1R3 deletion would reduce atherosclerotic plaque development in mice. We generated atherosclerotic mice with whole-body deletion of T1R3 by crossing T1R3-/- mice with ApoE-/- mice. T1R3+/+ ApoE-/- and T1R3-/- ApoE-/- mice were maintained on an atherogenic high-fat diet for 8 weeks. Weight gain and food consumption were measured during the 8-week diet. Atherosclerotic lesion development and size were assessed by en face analysis of intact aortas and microscopic analysis of aortic roots. Our results indicate that T1R3 deletion in male and female ApoE-/- mice reduces aortic atherosclerotic plaque accumulation. Hepatic triglyceride accumulation, which was measured by quantification of oil red O staining, was also reduced in T1R3-/- mice. While the ablation of T1R3 reduced the final body weight of both males and females by approximately 12%, serum lipids, insulin, and glucose were either unchanged or slightly reduced. Immunoblot analysis of the phosphorylation of p70S6K, an effector of mTORC1, suggests T1R3 ablation reduces mTORC1 activity by approximately 50% in the male livers. Collectively, these findings suggest that the whole-body deletion of T1R3 reduces atherosclerosis and hepatic steatosis in a manner largely independent of the measured effects on whole-body glucose and lipid homeostasis.

Regulation of beta adrenoceptor-mediated myocardial contraction and calcium dynamics by the G protein-coupled estrogen receptor 1.

The G protein-coupled estrogen receptor 1 (GPER) produces cardioprotective effects. However, the underlying mechanisms are not well understood. We aimed to investigate the role of GPER in ß adrenoceptor-mediated cardiac contraction and myocardial signaling. In anesthetized animals, intrajugular administration of isoproterenol produces a rapid and sustained rise in left ventricular pressure (LVP) and increases ectopic contractions. Administration of the GPER agonist G-1 during the plateau phase of isoproterenol-induced LVP increase rapidly restores LVP to baseline levels and reduces the frequency of ectopic contractions. In freshly isolated cardiomyocytes, isoproterenol potentiates electrically induced peak currents of L-type Ca2+ channels (LTCC) and increases the potential sensitivity of their inactivation. Coadministration of G-1 prevents isoproterenol-induced potentiation of peak LTCC currents and makes channels more sensitive to being inactivated compared to isoproterenol alone. Isoproterenol treatment of cardiomyocytes without electrical stimulation triggers slow-rising Ca2+ signals that are inhibited by the ß1AR antagonist metoprolol but not by ß2AR antagonist ICI-118551. G-1 pretreatment dose-dependently suppresses isoproterenol-induced total Ca2+ signals and the amplitude and frequency of the intrinsic Ca2+ oscillatory deflections. Pretreatment with the GPER antagonist G-36 produces opposite effects, dose-dependently increasing these signals. ISO promotes robust phosphorylation of Cav1.2 channels at Ser1928. G-1 pretreatment inhibits isoproterenol-stimulated phosphorylation of Cav1.2 at Ser1928, while G-36 pretreatment enhances this signal. Our data indicate that GPER functions as an intrinsic component of ß1AR signaling to moderate myocardial Ca2+ dynamics and contraction.

Loss of the nutrient sensor TAS1R3 leads to reduced bone resorption

The taste receptor type 1 (TAS1R) family of heterotrimeric G protein-coupled receptors participates in monitoring energy and nutrient status. TAS1R member 3 (TAS1R3) is a bi-functional protein that recognizes amino acids such as L-glycine and L-glutamate or sweet molecules such as sucrose and fructose when dimerized with TAS1R member 1 (TAS1R1) or TAS1R member 2 (TAS1R2), respectively. It was recently reported that deletion of TAS1R3 expression in Tas1R3 mutant mice leads to increased cortical bone mass but the underlying cellular mechanism leading to this phenotype remains unclear. Here, we independently corroborate the increased thickness of cortical bone in femurs of 20-week-old male Tas1R3 mutant mice and confirm that Tas1R3 is expressed in the bone environment. Tas1R3 is expressed in undifferentiated bone marrow stromal cells (BMSCs) in vitro and its expression is maintained during BMP2-induced osteogenic differentiation. However, levels of the bone formation marker procollagen type I N-terminal propeptide (PINP) are unchanged in the serum of 20-week-old Tas1R3 mutant mice as compared to controls. In contrast, levels of the bone resorption marker collagen type I C-telopeptide are reduced greater than 60% in Tas1R3 mutant mice. Consistent with this, Tas1R3 and its putative signaling partner Tas1R2 are expressed in primary osteoclasts and their expression levels positively correlate with differentiation status. Collectively, these findings suggest that high bone mass in Tas1R3 mutant mice is due to uncoupled bone remodeling with reduced osteoclast function and provide rationale for future experiments examining the cell-type-dependent role for TAS1R family members in nutrient sensing in postnatal bone remodeling

Loss of the nutrient sensor TAS1R3 leads to reduced bone resorption.

The taste receptor type 1 (TAS1R) family of heterotrimeric G protein-coupled receptors participates in monitoring energy and nutrient status. TAS1R member 3 (TAS1R3) is a bi-functional protein that recognizes amino acids such as L-glycine and L-glutamate or sweet molecules such as sucrose and fructose when dimerized with TAS1R member 1 (TAS1R1) or TAS1R member 2 (TAS1R2), respectively. It was recently reported that deletion of TAS1R3 expression in Tas1R3 mutant mice leads to increased cortical bone mass but the underlying cellular mechanism leading to this phenotype remains unclear. Here, we independently corroborate the increased thickness of cortical bone in femurs of 20-week-old male Tas1R3 mutant mice and confirm that Tas1R3 is expressed in the bone environment. Tas1R3 is expressed in undifferentiated bone marrow stromal cells (BMSCs) in vitro and its expression is maintained during BMP2-induced osteogenic differentiation. However, levels of the bone formation marker procollagen type I N-terminal propeptide (PINP) are unchanged in the serum of 20-week-old Tas1R3 mutant mice as compared to controls. In contrast, levels of the bone resorption marker collagen type I C-telopeptide are reduced greater than 60% in Tas1R3 mutant mice. Consistent with this, Tas1R3 and its putative signaling partner Tas1R2 are expressed in primary osteoclasts and their expression levels positively correlate with differentiation status. Collectively, these findings suggest that high bone mass in Tas1R3 mutant mice is due to uncoupled bone remodeling with reduced osteoclast function and provide rationale for future experiments examining the cell-type-dependent role for TAS1R family members in nutrient sensing in postnatal bone remodeling.

Kinase-mediated signaling cascades in mood disorders and antidepressant treatment.

Kinase-mediated signaling cascades regulate a number of different molecular mechanisms involved in cellular homeostasis, and are viewed as one of the most common intracellular processes that are robustly dysregulated in the pathophysiology of mood disorders such as depression. Newly emerged, rapid acting antidepressants are able to achieve therapeutic improvement, possibly in part, through stimulating activity of kinase-dependent signaling pathways. Thus, advancements in our understanding of how kinases may contribute to development and treatment of depression seem crucial. However, current investigations are limited to a single or small number of kinases and are unable to detect novel kinases. Here, we review fast developing kinome profiling approaches that allow identification of multiple kinases and kinase network connections simultaneously, analyze technical limitation and challenges, and discuss their future applications to mood disorders and antidepressant treatment.

Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells.

The MAPKs ERK1/2 respond to nutrients and other insulin secretagogues in pancreatic ß-cells and mediate nutrient-dependent insulin gene transcription. Nutrients also stimulate the mechanistic target of rapamycin complex 1 (mTORC1) to regulate protein synthesis. We showed previously that activation of both ERK1/2 and mTORC1 in the MIN6 pancreatic ß-cell-derived line by extracellular amino acids (AAs) is at least in part mediated by the heterodimeric T1R1/T1R3, a G protein-coupled receptor. We show here that AAs differentially activate these two signaling pathways in MIN6 cells. Pretreatment with pertussis toxin did not prevent the activation of either ERK1/2 or mTORC1 by AAs, indicating that G(I) is not central to either pathway. Although glucagon-like peptide 1, an agonist for a G(s-)coupled receptor, activated ERK1/2 well and mTORC1 to a small extent, AAs had no effect on cytosolic cAMP accumulation. Ca(2+) entry is required for ERK1/2 activation by AAs but is dispensable for AA activation of mTORC1. Pretreatment with UBO-QIC, a selective G(q) inhibitor, reduced the activation of ERK1/2 but had little effect on the activation of mTORC1 by AAs, suggesting a differential requirement for G(q). Inhibition of G(12/13) by the overexpression of the regulator of G protein signaling domain of p115 ρ-guanine nucleotide exchange factor had no effect on mTORC1 activation by AAs, suggesting that these G proteins are also not involved. We conclude that AAs regulate ERK1/2 and mTORC1 through distinct signaling pathways.

G protein-coupled receptors and the regulation of autophagy.

Autophagy is an important catabolic cellular process that eliminates damaged and unnecessary cytoplasmic proteins and organelles. Basal autophagy occurs during normal physiological conditions, but the activity of this process can be significantly altered in human diseases. Thus, defining the regulatory inputs and signals that control autophagy is essential. Nutrients are key modulators of autophagy. Although autophagy is generally accepted to be regulated in a cell-autonomous fashion, recent studies suggest that nutrients can modulate autophagy in a systemic manner by inducing the secretion of hormones and neurotransmitters that regulate G protein-coupled receptors (GPCRs). Emerging studies show that GPCRs also regulate autophagy by directly detecting extracellular nutrients. We review the role of GPCRs in autophagy regulation, highlighting their potential as therapeutic drug targets.

Muscarinic control of MIN6 pancreatic ß cells is enhanced by impaired amino acid signaling.

We have shown recently that the class C G protein-coupled receptor T1R1/T1R3 taste receptor complex is an early amino acid sensor in MIN6 pancreatic ß cells. Amino acids are unable to activate ERK1/2 in ß cells in which T1R3 has been depleted. The muscarinic receptor agonist carbachol activated ERK1/2 better in T1R3-depleted cells than in control cells. Ligands that activate certain G protein-coupled receptors in pancreatic ß cells potentiate glucose-stimulated insulin secretion. Among these is the M3 muscarinic acetylcholine receptor, the major muscarinic receptor in ß cells. We found that expression of M3 receptors increased in T1R3-depleted MIN6 cells and that calcium responses were altered. To determine whether these changes were related to impaired amino acid signaling, we compared responses in cells exposed to reduced amino acid concentrations. M3 receptor expression was increased, and some, but not all, changes in calcium signaling were mimicked. These findings suggest that M3 acetylcholine receptors are increased in ß cells as a mechanism to compensate for amino acid deficiency.

Minireview: Nutrient sensing by G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are membrane proteins that recognize molecules in the extracellular milieu and transmit signals inside cells to regulate their behaviors. Ligands for many GPCRs are hormones or neurotransmitters that direct coordinated, stereotyped adaptive responses. Ligands for other GPCRs provide information to cells about the extracellular environment. Such information facilitates context-specific decision making that may be cell autonomous. Among ligands that are important for cellular decisions are amino acids, required for continued protein synthesis, as metabolic starting materials and energy sources. Amino acids are detected by a number of class C GPCRs. One cluster of amino acid-sensing class C GPCRs includes umami and sweet taste receptors, GPRC6A, and the calcium-sensing receptor. We have recently found that the umami taste receptor heterodimer T1R1/T1R3 is a sensor of amino acid availability that regulates the activity of the mammalian target of rapamycin. This review focuses on an array of findings on sensing amino acids and sweet molecules outside of neurons by this cluster of class C GPCRs and some of the physiologic processes regulated by them.

Off-target effects of MEK inhibitors.

The mitogen-activated protein kinases (MAPKs) ERK1/2 regulate numerous cellular processes, including gene transcription, proliferation, and differentiation. The only known substrates of the MAP2Ks MEK1/2 are ERK1/2; thus, MEK inhibitors PD98059, U0126, and PD0325901 have been important tools in determining the functions of ERK1/2. By using these inhibitors and genetically manipulating MEK, we found that ERK1/2 activation is neither sufficient nor necessary for regulated secretion of insulin from pancreatic ß cells or secretion of epinephrine from chromaffin cells. We show that both PD98059 and U0126 reduce agonist-induced entry of calcium into cells in a manner independent of their ability to inhibit ERK1/2. Caution should be used when interpreting results from experiments using these compounds.

Amino acid regulation of autophagy through the GPCR TAS1R1-TAS1R3.

Cells require the ability to rapidly detect decreases in concentrations of free amino acids so that homeostatic mechanisms, including autophagy, can be engaged to replenish amino acids. Amino acids are transported into cells where it is generally accepted that they are detected by an intracellular sensor. We now show that the cell surface G protein coupled receptor (GPCR) TAS1R1-TAS1R3 (T1R1-T1R3) can sense extracellular amino acids, activate MTORC1, and inhibit autophagy. This receptor is expressed in most tissues and fasted TAS1R3 (-/-) mice have increased autophagy in the heart, skeletal muscle and liver.

The G protein-coupled taste receptor T1R1/T1R3 regulates mTORC1 and autophagy.

Cells continually assess their energy and nutrient state to maintain growth and survival and engage necessary homeostatic mechanisms. Cell-autonomous responses to the fed state require the surveillance of the availability of amino acids and other nutrients. The mammalian target of rapamycin complex 1 (mTORC1) integrates information on nutrient and amino acid availability to support protein synthesis and cell growth. We identify the G protein-coupled receptor (GPCR) T1R1/T1R3 as a direct sensor of the fed state and amino acid availability. Knocking down this receptor, which is found in most tissues, reduces the ability of amino acids to signal to mTORC1. Interfering with this receptor alters localization of mTORC1, downregulates expression of pathway inhibitors, upregulates key amino acid transporters, blocks translation initiation, and induces autophagy. These findings reveal a mechanism for communicating amino acid availability through a GPCR to mTORC1 in mammals.

RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly.

The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. We find that the Ras-like small G protein, RalB, is localized to nascent autophagosomes and is activated on nutrient deprivation. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery.

MAP-ping unconventional protein-DNA interactions.

Control of gene expression depends on a myriad of protein-DNA interactions, and the number of proteins involved just got larger. In this issue, Hu et al. (2009) identify hundreds of human proteins that bind to DNA, including many surprises such as the protein kinase ERK2 (MAPK1) that now appears to control gene expression directly.

The human Rad9 checkpoint protein stimulates the carbamoyl phosphate synthetase activity of the multifunctional protein CAD.

The human Rad9 checkpoint protein is a subunit of the heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex that plays a role as a damage sensor in the DNA damage checkpoint response. Rad9 has been found to interact with several other proteins outside the context of the 9-1-1 complex with no obvious checkpoint functions. During our studies on the 9-1-1 complex, we found that Rad9 immunoprecipitates contained a 240 kDa protein that was identified as carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD), a multienzymatic protein required for the de novo synthesis of pyrimidine nucleotides and cell growth. Further investigations revealed that only free Rad9, but not Rad9 within the 9-1-1 complex, bound to CAD. The rate-limiting step in de novo pyrimidine nucleotide synthesis is catalyzed by the carbamoyl phosphate synthetase II (CPSase) domain of CAD. We find that Rad9 binds to the CPSase domain, and, moreover, this binding results in a 2-fold stimulation of the CPSase activity of CAD. Similar results were also obtained with an N-terminal Rad9 fragment. These findings suggest that Rad9 may play a role in ribonucleotide biosynthesis.

Sodium arsenite inhibits and reverses expression of adipogenic and fat cell-specific genes during in vitro adipogenesis.

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 p21(Cip1/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 p21(Cip1/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.