The Loss of PPARγ Expression and Signaling Is a Key Feature of Cutaneous Actinic Disease and Squamous Cell Carcinoma: Association with Tumor Stromal Inflammation.

Given the importance of peroxisome proliferator-activated receptor (PPAR)-gamma in epidermal inflammation and carcinogenesis, we analyzed the transcriptomic changes observed in epidermal PPARγ-deficient mice (Pparg-/-epi). A gene set enrichment analysis revealed a close association with epithelial malignancy, inflammatory cell chemotaxis, and cell survival. Single-cell sequencing of Pparg-/-epi mice verified changes to the stromal compartment, including increased inflammatory cell infiltrates, particularly neutrophils, and an increase in fibroblasts expressing myofibroblast marker genes. A comparison of transcriptomic data from Pparg-/-epi and publicly available human and/or mouse actinic keratoses (AKs) and cutaneous squamous cell carcinomas (SCCs) revealed a strong correlation between the datasets. Importantly, PPAR signaling was the top common inhibited canonical pathway in AKs and SCCs. Both AKs and SCCs also had significantly reduced PPARG expression and PPARγ activity z-scores. Smaller reductions in PPARA expression and PPARα activity and increased PPARD expression but reduced PPARδ activation were also observed. Reduced PPAR activity was also associated with reduced PPARα/RXRα activity, while LPS/IL1-mediated inhibition of RXR activity was significantly activated in the tumor datasets. Notably, these changes were not observed in normal sun-exposed skin relative to non-exposed skin. Finally, Ppara and Pparg were heavily expressed in sebocytes, while Ppard was highly expressed in myofibroblasts, suggesting that PPARδ has a role in myofibroblast differentiation. In conclusion, these data provide strong evidence that PPARγ and possibly PPARα represent key tumor suppressors by acting as master inhibitors of the inflammatory changes found in AKs and SCCs.

Epidermal PPARγ Is a Key Homeostatic Regulator of Cutaneous Inflammation and Barrier Function in Mouse Skin.

Both agonist studies and loss-of-function models indicate that PPARγ plays an important role in cutaneous biology. Since PPARγ has a high level of basal activity, we hypothesized that epidermal PPARγ would regulate normal homeostatic processes within the epidermis. In this current study, we performed mRNA sequencing and differential expression analysis of epidermal scrapings from knockout mice and wildtype littermates. Pparg-/-epi mice exhibited a 1.5-fold or greater change in the expression of 11.8% of 14,482 identified transcripts. Up-regulated transcripts included those for a large number of cytokines/chemokines and their receptors, as well as genes associated with inflammasome activation and keratinization. Several of the most dramatically up-regulated pro-inflammatory genes in Pparg-/-epi mouse skin included Igfl3, 2610528A11Rik, and Il1f6. RT-PCR was performed from RNA obtained from non-lesional full-thickness skin and verified a marked increase in these transcripts, as well as transcripts for Igflr1, which encodes the receptor for Igfl3, and the 2610528A11Rik receptor (Gpr15). Transcripts for Il4 were detected in Pparg-/-epi mouse skin, but transcripts for Il17 and Il22 were not detected. Down-regulated transcripts included sebaceous gland markers and a number of genes associated with lipid barrier formation. The change in these transcripts correlates with an asebia phenotype, increased transepidermal water loss, alopecia, dandruff, and the appearance of spontaneous inflammatory skin lesions. Histologically, non-lesional skin showed hyperkeratosis, while inflammatory lesions were characterized by dermal inflammation and epidermal acanthosis, spongiosis, and parakeratosis. In conclusion, loss of epidermal Pparg alters a substantial set of genes that are associated with cutaneous inflammation, keratinization, and sebaceous gland function. The data indicate that epidermal PPARγ plays an important role in homeostatic epidermal function, particularly epidermal differentiation, barrier function, sebaceous gland development and function, and inflammatory signaling.

Evidence for a non-stochastic two-field hypothesis for persistent skin cancer risk.

With recurring carcinogen exposures, individual tumors develop in a field of genetic mutations through a stepwise process of clonal expansion and evolution. Once established, this "cancer field" persists in the absence of continued carcinogen exposures, resulting in a sustained risk for cancer development. Using a bioimaging approach, we previously demonstrated that a dermal premalignant field characterized by inflammatory angiogenesis persists following the cessation of ultraviolet light exposures and accurately predicts future overlying epidermal tumor formation. Following ultraviolet light treatments, others have observed that patches of p53 immunopositive cells persist stochastically throughout the epidermal stem cell population. However, these studies were done by random biopsies, introducing sampling bias. We now show that, rather than being randomly distributed, p53+ epidermal cells are enriched only in areas overlying this multi-focal dermal field. Moreover, we also show that the dermal field is characterized by a senescent phenotype. We propose that persistence of the overlying epithelial cancerization field in the absence of exogenous carcinogens or promoters requires a two-field composite consisting of a dermal senescent field driving the persistence of the overlying epidermal cancer field. These observations challenge current models that suggest that persistence of cancer risk in the absence of continued carcinogen exposures is simply a function of stochastically arranged, long-lived but dormant epithelial clonal stem cells mutants. The model proposed here could provide new insights into how cancer risk persists following cessation of carcinogenic exposures.

The PPARγ Agonist Rosiglitazone Suppresses Syngeneic Mouse SCC (Squamous Cell Carcinoma) Tumor Growth through an Immune-Mediated Mechanism.

Recent evidence suggests that PPARγ agonists may promote anti-tumor immunity. We show that immunogenic PDV cutaneous squamous cell carcinoma (CSCC) tumors are rejected when injected intradermally at a low cell number (1 × 106) into immune competent syngeneic hosts, but not immune deficient mice. At higher cell numbers (5 × 106 PDV cells), progressively growing tumors were established in 14 of 15 vehicle treated mice while treatment of mice with the PPARγ agonist rosiglitazone resulted in increased tumor rejection (5 of 14 tumors), a significant decrease in PDV tumor size, and a significant decrease in tumor cell Ki67 labeling. Rosiglitazone treatment had no effect on tumor rejection, tumor volume or PDV tumor cell proliferation in immune deficient NOD.CB17-PrkdcSCID/J mice. Rosiglitazone treatment also promoted an increase in tumor infiltrating CD3+ T-cells at both early and late time points. In contrast, rosiglitazone treatment had no significant effect on myeloid cells expressing either CD11b or Gr-1 but suppressed a late accumulation of myeloid cells expressing both CD11b and Gr-1, suggesting a potential role for CD11b+Gr-1+ myeloid cells in the late anti-tumor immune response. Overall, our data provides evidence that the PPARγ agonist rosiglitazone promotes immune-mediated anti-neoplastic activity against tumors derived from this immunogenic CSCC cell line.

Epidermal PPARγ influences subcutaneous tumor growth and acts through TNF-α to regulate contact hypersensitivity and the acute photoresponse.

It is known that ultraviolet B (UVB) induces PPARγ ligand formation while loss of murine epidermal PPARγ (Pparg-/-epi) promotes UVB-induced apoptosis, inflammation, and carcinogenesis. PPARγ is known to suppress tumor necrosis factor-α (TNF-α) production. TNF-α is also known to promote UVB-induced inflammation, apoptosis, and immunosuppression. We show that Pparg-/-epi mice exhibit increased baseline TNF-α expression. Neutralizing Abs to TNF-α block the increased photo-inflammation and photo-toxicity that is observed in Pparg-/-epi mouse skin. Interestingly, the increase in UVB-induced apoptosis in Pparg-/-epi mice is not accompanied by a change in cyclobutane pyrimidine dimer clearance or in mutation burden. This suggests that loss of epidermal PPARγ does not result in a significant alteration in DNA repair capacity. However, loss of epidermal PPARγ results in marked immunosuppression using a contact hypersensitivity (CHS) model. This impaired CHS response was significantly alleviated using neutralizing TNF-α antibodies or loss of germline Tnf. In addition, the PPARγ agonist rosiglitazone reversed UVB-induced systemic immunosuppression (UV-IS) as well as UV-induced growth of B16F10 melanoma tumor cells in syngeneic mice. Finally, increased B16F10 tumor growth was observed when injected subcutaneously into Pparg-/-epi mice. Thus, we provide novel evidence that epidermal PPARγ is important for cutaneous immune function and the acute photoresponse.

Comparison of the acute ultraviolet photoresponse in congenic albino hairless C57BL/6J mice relative to outbred SKH1 hairless mice.

Hairless albino Crl:SKH1-Hr(hr) mice are commonly utilized for studies in which hair or pigmentation would introduce an impediment to observational studies. Being an outbred strain, the SKH1 model suffers from key limitations that are not seen with congenic mouse strains. Inbred and congenic C57BL/6J mice are commonly utilized for modified genetic mouse models. We compare the acute UV-induced photoresponse between outbred SKH1 mice and an immune competent, hairless, albino C57BL/6J congenic mouse line [B6.Cg-Tyr(c-2J) Hr(hr) /J]. Histologically, B6.Cg-Tyr(c-2J) Hr(hr) /J skin is indistinguishable from that of SKH1 mice. The skin of both SKH1 and B6.Cg-Tyr(c-2J) Hr(hr) /J mice exhibited a reduction in hypodermal adipose tissue, the presence of utricles and dermal cystic structures, the presence of dermal granulomas and epidermal thickening. In response to a single 1500 J/m(2) ultraviolet B dose, the oedema and apoptotic responses were equivalent in both mouse strains. However, B6.Cg-Tyr(c-2J) Hr(hr) /J mice exhibited a more robust delayed sunburn reaction, with an increase in epidermal erosion, scab formation and myeloperoxidase activity relative to SKH1 mice. Compared with SKH1 mice, B6.Cg-Tyr(c-2J) Hr(hr) /J also exhibited an aberrant proliferative response to this single UV exposure. Epidermal Ki67 immunopositivity was significantly suppressed in B6.Cg-Tyr(c-2J) Hr(hr) /J mice at 24 h post-UV. A smaller non-significant reduction in Ki67 labelling was observed in SKH1 mice. Finally, at 72 h post-UV, SKH1 mice, but not B6.Cg-Tyr(c-2J) Hr(hr) /J mice, exhibited a significant increase in Ki67 immunolabelling relative to non-irradiated controls. Thus, B6.Cg-Tyr(c-2J) Hr(hr) /J mice are suitable for photobiology experiments.

Gestational diabetes induces alterations in the function of neonatal endothelial colony-forming cells.

BACKGROUND: Children born to mothers with gestational diabetes mellitus (GDM) experience increased risk of developing hypertension, type 2 diabetes mellitus, and obesity. Disrupted function of endothelial colony-forming cells (ECFCs) may contribute to this enhanced risk. The goal of this study was to determine whether cord blood ECFCs from GDM pregnancies exhibit altered functionality. METHODS: ECFCs isolated from the cord blood of control and GDM pregnancies were assessed for proliferation, senescence, and Matrigel network formation. The requirement for p38MAPK in hyperglycemia-induced senescence was determined using inhibition and overexpression studies. RESULTS: GDM-exposed ECFCs were more proliferative than control ECFCs. However, GDM-exposed ECFCs exhibited decreased network-forming ability in Matrigel. Aging of ECFCs by serial passaging led to increased senescence and reduced proliferation of GDM-exposed ECFCs. ECFCs from GDM pregnancies were resistant to hyperglycemia-induced senescence compared with those from controls. In response to hyperglycemia, control ECFCs activated p38MAPK, which was required for hyperglycemia-induced senescence. In contrast, GDM-exposed ECFCs showed no change in p38MAPK activation under equivalent conditions. CONCLUSION: Intrauterine exposure of ECFCs to GDM induces unique phenotypic alterations. The resistance of GDM-exposed ECFCs to hyperglycemia-induced senescence and decreased p38MAPK activation suggest that these progenitor cells have undergone changes that induce tolerance to a hyperglycemic environment.

Impaired function of Fanconi anemia type C-deficient macrophages.

FA is a genetic disorder characterized by BM failure, developmental defects, and cancer predisposition. Previous studies suggest that FA patients exhibit alterations in immunologic function. However, it is unclear whether the defects are immune cell-autonomous or secondary to leukopenia from evolving BM failure. Given the central role that macrophages have in the innate immune response, inflammation resolution, and antigen presentation for acquired immunity, we examined whether macrophages from Fancc-/- mice exhibit impaired function. Peritoneal inflammation induced by LPS or sodium periodate resulted in reduced monocyte/macrophage recruitment in Fancc-/- mice compared with WT controls. Fancc-/- mice also had decreased inflammatory monocytes mobilized into the peripheral blood after LPS treatment compared with controls. Furthermore, Fancc-/- peritoneal macrophages displayed cell-autonomous defects in function, including impaired adhesion to FN or endothelial cells, reduced chemoattractant-mediated migration, and decreased phagocytosis. Moreover, dysregulated F-actin rearrangement was detected in Fancc-/- macrophages after adhesion to FN, which was consistent with an observed reduction in RhoA-GTP levels. Importantly, these data suggest that impaired cytoskeletal rearrangements in Fancc-/- macrophages may be the common mechanism responsible for cell-autonomous defects detected in vitro, as well as altered monocyte/macrophage trafficking in vivo.

p21-activated kinase regulates mast cell degranulation via effects on calcium mobilization and cytoskeletal dynamics.

Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (FcepsilonRI) resulting in release of proinflammatory mediators. The biochemical pathways linking IgE activation to calcium influx and cytoskeletal changes required for intracellular granule release are incompletely understood. We demonstrate, genetically, that Pak1 is required for this process. In a passive cutaneous anaphylaxis experiment, W(sh)/W(sh) mast cell-deficient mice locally reconstituted with Pak1(-/-) bone marrow-derived mast cells (BMMCs) experienced strikingly decreased allergen-induced vascular permeability compared with controls. Consistent with the in vivo phenotype, Pak1(-/-) BMMCs exhibited a reduction in FcepsilonRI-induced degranulation. Further, Pak1(-/-) BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to FcepsilonRI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases.

In vitro hyperglycemia or a diabetic intrauterine environment reduces neonatal endothelial colony-forming cell numbers and function.

OBJECTIVE: Emerging data demonstrate that maternal diabetes has long-term health consequences for offspring, including the development of hypertension. In adults, circulating endothelial progenitor cells (EPCs) participate in vascular repair, and EPC numbers and function inversely correlate with the risk of developing vascular disease. Therefore, our objectives were to determine whether hyperglycemia or exposure to a diabetic intrauterine environment alters EPC function. RESEARCH DESIGN AND METHODS: We used well-established clonogenic endothelial colony-forming cell (ECFC) assays and murine transplantation experiments to examine human vasculogenesis. RESULTS: Both in vitro hyperglycemia and a diabetic intrauterine environment reduced ECFC colony formation, self-renewal capacity, and capillary-like tube formation in matrigel. This cellular phenotype was linked to premature senescence and reduced proliferation. Further, cord blood ECFCs from diabetic pregnancies formed fewer chimeric vessels de novo after transplantation into immunodeficient mice compared with neonatal ECFCs harvested from uncomplicated pregnancies. CONCLUSIONS; Collectively, these data demonstrate that hyperglycemia or exposure to a diabetic intrauterine environment diminishes neonatal ECFC function both in vitro and in vivo, providing potential mechanistic insights into the long-term cardiovascular complications observed in newborns of diabetic pregnancies.

Alterations in brain protein kinase C isoforms following developmental exposure to a polychlorinated biphenyl mixture.

PCBs have been shown to alter several neurochemical end-points and are implicated in the etiology of some neurological diseases. Recent in vivo studies from our laboratory indicated that developmental exposure to a commercial PCB mixture, Aroclor 1254, caused perturbations in calcium homeostasis and changes in protein kinase C (PKC) activities in rat brain. However, it is not known which molecular substances are targets for PCB-induced developmental neurotoxicity. Since the PKC signaling pathway has been implicated in the modulation of motor behavior as well as learning and memory, and the roles of PKC are subspecies specific, the present study attempted to analyze the effects on selected PKC isozymes in the cerebellum and the hippocampus following developmental exposure (gestational day 6 through postnatal day 21) to a PCB mixture, Aroclor 1254. The results indicated that the developmental exposure to PCBs caused significant hypothyroxinemia and age-dependent alterations in the translocation of PKC isozymes; the effects were greatly significant at postnatal day (PND) 14. Immunoblot analysis of PKC-alpha (alpha) from both cerebellum and hippocampus revealed that developmental exposure to Aroclor 1254 caused a significant decrease in cytosolic fraction and an increase in particulate fraction. There was no significant difference between these two brain regions on the level of fractional changes. However, the ratio between the fractions (particulate/cytosol) from cerebellum only was increased in a dose-dependent manner. Analysis of PKC-gamma (gamma) in cerebellum on PND14 showed a decrease in cytosolic fraction in both dose groups and an increase in particulate fraction at high dose (6 mg/kg) only. The ratio between the two fractions was increased in a dose-dependent manner. In the hippocampus, there was a significant decrease in PKC-gamma in cytosolic fraction of the high-dose group and a significant increase in particulate fraction of the low-dose group. But, the ratio between the fractions showed a significant increase (2.6-fold increase in high dose on PND14). Analysis of PKC-epsilon (epsilon) in cerebellum showed a significant decrease in cytosolic fraction at PND14, while particulate PKand an increase in ratio between fractions at 6 mg/kg on PND14. The results from this study indicate that the patterns of subcellular distributions of PKC isoforms following a developmental PCB exposure were PKC isozyme- and developmental stage-specific. Considering the significant role of PKC signaling in motor behavior, learning and memory, it is suggested that altered subcellular distribution of PKC isoforms at critical periods of brain development may be a possible mechanism of PCB-induced neurotoxic effects and that PKC-alpha, gamma, and epsilon may be among the target molecules implicated with PCB-induced neurological impairments during developmental exposure. It is believed that this is the first report successfully identifying PKC isoforms responding to PCBs during developmental exposure.

Increased [3H]phorbol ester binding in rat cerebellar granule cells and inhibition of 45Ca(2+) buffering in rat cerebellum by hydroxylated polychlorinated biphenyls.

Our previous structure-activity relationship (SAR) studies indicated that the effects of polychlorinated biphenyls (PCBs) on neuronal Ca(2+) homeostasis and protein kinase C (PKC) translocation were associated with the extent of coplanarity. Chlorine substitutions at ortho position on the biphenyl, which increase the non-coplanarity, are characteristic of the most active congeners in vitro. In the present study, we investigated the effects of selected hydroxylated PCBs, which are major PCB metabolites identified in mammals, on the same measures where PCBs had differential effects based on structural configuration. These measures include PKC translocation as determined by [3H]phorbol ester ([3H]PDBu) binding in cerebellar granule cells, and Ca(2+) sequestration as determined by 45Ca(2+) uptake by microsomes isolated from adult rat cerebellum. All the selected hydroxy-PCBs with ortho-chlorine substitutions increased [3H]PDBu binding in a concentration-dependent manner and the order of potency as determined by E(50) (concentration that increases control activity by 50%) is 2',4',6'-trichloro-4-biphenylol (32 +/- 4 microM), 2',5'-dichloro-4-biphenylol (70 +/- 9 microM), 2,2',4',5,5'-pentachloro-4-biphenylol (80 +/- 7 microM) and 2,2',5'-trichloro-4-biphenylol (93 +/- 14 microM). All the selected hydroxy-PCBs inhibited microsomal 45Ca(2+) uptake to a different extent. Among the hydroxy-PCBs selected, 2',4',6'-trichloro-4-biphenylol is the most active in increasing [3H]PDBu binding as well as inhibiting microsomal 45Ca(2+) uptake. 3,5-Dichloro-4-biphenylol and 3,4',5-trichloro-4-biphenylol did not increase [3H]PDBu binding, but inhibited microsomal 45Ca(2+) uptake. This effect was not related to ionization of these two hydroxy-PCBs. Hydroxylated PCBs seemed to be as active as parent PCBs in vitro. These studies indicate that PCB metabolites such as hydroxy-PCBs might contribute significantly to the neurotoxic responses of PCBs.

Differential effects of polybrominated diphenyl ethers and polychlorinated biphenyls on [3H]arachidonic acid release in rat cerebellar granule neurons.

Polybrominated diphenyl ethers (PBDEs), which are widely used as flame-retardants, have been increasing in environmental and human tissue samples during the past 20-30 years, while other structurally related, persistent organic pollutants such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins (on a TEQ basis), have decreased. PBDEs have been detected in human blood, adipose tissue, and breast milk, and developmental and long-term exposure to these contaminants may pose a human health risk, especially to children. Previously, we demonstrated that PCBs, which cause neurotoxic effects, including changes in learning and memory, stimulated the release of [(3)H]arachidonic acid ([(3)H]AA) by a cPLA(2)/iPLA(2)-dependent mechanism. PLA(2)(phospholipase A(2)) activity has been associated with learning and memory, and AA has been identified as a second messenger involved in synaptic plasticity. The objective of the present study was to test whether PBDE mixtures (DE-71 and DE-79), like other organohalogen mixtures, have a similar action on [(3)H]AA release in an in vitro neuronal culture model. Cerebellar granule cells at 7 days in culture were labeled with [(3)H]AA for 16-20 h and then exposed in vitro to PBDEs. DE-71, a mostly pentabromodiphenyl ether mixture, significantly stimulated [(3)H]AA release at concentrations as low as 10 microg/ml, while DE-79, a mostly octabromodiphenyl ether mixture, did not stimulate [(3)H]AA release, even at 50 microg/ml. The release of [(3)H]AA by DE-71 is time-dependent, and a significant increase was seen after only 5-10 min of exposure. The removal and chelation of calcium from the exposure buffer, using 0.3 mM EGTA, significantly attenuated the DE-71-stimulated [(3)H]AA release; however, only an 18% inhibition of the release was demonstrated for the calcium replete conditions at 30 microg/ml DE-71. Methyl arachidonylfluorophosphonate (5 microM), an inhibitor of cPLA(2)/iPLA(2), completely attenuated the DE-71-stimulated [(3)H]AA release. Further studies focused on comparing the effects of DE-71 with PCB mixtures such as Aroclors 1016 and 1254. Both PCB mixtures stimulated [(3)H]AA release in a concentration-dependent manner; however, the effect for PCBs was about two times greater than that of the PBDEs on a weight basis, but was comparable on a molar basis. These results indicate that PBDEs stimulated the release of [(3)H]AA by activating PLA(2), which is similar to the effect of other organohalogen mixtures.

Neurobehavioral assessments of rats perinatally exposed to a commercial mixture of polychlorinated biphenyls.

Because of behavioral deficits associated with gestational exposure to PCBs in children, we sought to quantify neurobehavioral effects of perinatal exposure to Aroclor 1254(R) (A1254), a commercial mixture of PCBs, in rats. Pregnant Long-Evans rats were fed A1254 at doses of 0, 1.0, or 6.0 mg/kg/day throughout gestation and nursing. The growth and behavior of their male and female offspring were assessed both during development and as adults, using a variety of behavioral tests that included a neurobehavioral screening battery (functional observational battery [FOB] and automated tests of locomotor activity), habituation of motor activity, acquisition of a visual discrimination, and performance of a visual signal-detection task. During the suckling period, A1254 at 6 mg/kg reduced survival and body weight gain of offspring of both sexes; however, locomotor activity was unaffected, and only small and transient changes in other measures were evident. In adulthood, perinatal exposure to A1254 did not affect habituation of locomotor activity, acquisition of the visual discrimination, or sustained attention. Rats performing the signal-detection task were challenged with cocaine (0, 1.25, 2.5, 5.0 mg/kg) and haloperidol (0, 0.003, 0.010, 0.030 mg/kg) to probe the integrity of dopaminergic systems in the central nervous system (CNS). A1254 did not alter the impairment of attention caused by haloperidol. Cocaine reduced false alarms more in controls than in rats exposed to A1254, but the effect was not clearly related to the dose of A1254. Perinatal exposure to this commercial PCB mixture had very little effect on these tests of behavior during development and in adulthood.