BACKGROUND

Lichen planus (LP) is a chronic common mucocutaneous inflammatory disorder of uncertain aetiology. An association between hepatitis C virus (HCV) infection and LP has been recognised, particularly in Italy, Spain and Japan. The pathogenesis of such an association is unclear, but it may be due to cell-mediated cytotoxicity to an epitope shared by HCV and damaged keratinocytes. Recent studies using in situ hybridization suggest that HCV may replicate in the oral mucosa.

OBJECTIVE

The aim of the present study was to examine the oral epithelium of patients with oral LP for evidence of HCV-RNA by polymerase chain reaction (PCR) and to examine the relationship to cytokines including interferon (INF-gamma), interleukins (IL-1, IL-2, IL-4, IL-6, IL-8 , and IL-10), tumour necrosis factor (TNF-alpha) and transforming growth factor (TGFbeta-1).

METHODS

We selected 100 Italian patients, and divided them into 4 groups. Group A consisted of 25 HCV+ve patients with erosive oral LP. Group B was a control group constituted by 25 healthy HCV -ve subjects with no LP. Group C consisted of 25 HCV-ve patients with oral reticular LP and Group D was made of 25 HCV-ve patients with oral erosive LP. The patients of group A (test group) were submitted to oral biopsy with 2 samples of epithelium, lesional and non-lesional, and a 10 ml peripheral blood sample was taken. The patients of group B (negative control), C and D (comparison groups) were submitted to oral epithelial biopsy and a 10 ml peripheral blood sample was collected. PCR was used to search for HCV-RNA in biopsy material. Cytokines INF-gamma ,IL-1, IL-2, IL-4, IL-6, IL-8 , IL-10 and TNF-alpha and TGFbeta-1 were assayed in serum.

CONCLUSIONS

PCR did not detect the viral genome in oral epithelium of the patients with oral LP and HCV+ve (group A), but there was an increase in levels of TNF-alpha and a reduction of IL-1, INF-gamma and IL-8 compared to patients who had oral reticular LP but HCV-ve and to patients who had oral erosive LP but HCV-ve, and compared to negative controls. The results indicate that patients of group A showed a reduction of pro-inflammatory but an increase in immunomodulant cytokines. The results suggest the possibility that HCV exerts an indirect effect, mediated possibly by the induction of cytokines and lymphokines.

Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) promotes angiogenesis and plays important roles both in physiological and pathological conditions. VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and are originally considered specific to endothelial cells. We previously reported that VEGFRs were also constitutively expressed in normal human <em>keratinocytes</em> and overexpressed in psoriatic epidermis. In addition, UVB can activate VEGFRs in normal <em>keratinocytes</em>, and the activated VEGFR-<em>2</em> signaling is involved in the pro-survival mechanism. Here, we show that VEGFRs were also upregulated and activated by UVA in normal human <em>keratinocytes</em> via PKC, and interestingly, both the activated VEGFR-1 and VEGFR-<em>2</em> protected against UVA-induced cell death. As VEGFRs were over-expressed in psoriatic epidermis, we further investigated whether narrowband UVB (NB-UVB) phototherapy or topical halomethasone monohydrate 0.05% cream could affect their expression. Surprisingly, the over-expressed VEGFRs in psoriatic epidermis were significantly attenuated by both treatments. During NB-UVB therapy, VEGFRs declined first in the basal, and then gradually in the upper psoriatic epidermis. VEGFRs were activated in psoriatic epidermis, their activation was enhanced by NB-UVB, but turned undetectable after whole therapy. This process was quite different from that by halomethasone, in which VEGFRs and phospho-VEGFRs decreased in a gradual, homogeneous manner. Our findings further suggest that UV-induced activation of VEGFRs serves as a pro-survival signal for <em>keratinocytes</em>. In addition, VEGFRs may be involved in the pathological process of psoriasis, and UV phototherapy is effective for psoriasis by directly modulating the expression of VEGFRs.

We have examined the <em>growth</em> of three epitheliotropic viruses in cultures of human epidermal <em>keratinocytes</em>: herpes simplex virus (HSV) type 1, adenovirus type <em>2</em> (Ad-<em>2</em>), and human papillomavirus (HPV) type 1. Differences were noted in the level of expression of each virus, and these differences may be related to a dependency or lack of dependency on <em>keratinocyte</em> differentiation for complete viral <em>growth</em>. Of the three viruses studied, HSV was the only one to replicate productively in all cells of the culture. Its expression was independent of <em>keratinocyte</em> differentiation. This is unlike Ad-<em>2</em>, which infected all cells in the culture but replicated productively only in the suprabasal cells. Basal <em>keratinocytes</em> were shown to be infected, but for unknown reasons, they appeared in most instances to be nonpermissive for Ad-<em>2</em> replication. Infected basal <em>keratinocytes</em> became permissive when they reached a suprabasal position. Ad-<em>2</em> appears to require <em>keratinocyte</em> differentiation for full expression in culture. Following infection with HPV, cultured <em>keratinocytes</em> showed no evidence of productive replication. However, 50 to <em>2</em>50 copies of HPV DNA could be detected in each cell (average) as stable nonintegrated molecules. Viral DNA replication has been shown to occur in the younger cells and not in the older, more differentiated <em>keratinocytes</em>. The failure of HPV to be fully expressed in culture may be related, in part, to incomplete differentiation of the <em>keratinocyte</em> in vitro. The major conclusions of this study are (1) that <em>keratinocyte</em> differentiation is likely to play a role in the expression of some epitheliotropic viruses in culture, and (<em>2</em>) that <em>keratinocyte</em> differentiation may be a <em>factor</em> in the pathogenesis of certain viral diseases of keratinizing epithelia.

During <em>keratinocyte</em> differentiation, the glycolipid, glucosylceramide (GlcCer), is thought to be synthesized, stored in intracellular lamellar granules and eventually extruded into the intercellular space where GlcCer is hydrolyzed to ceramide, a major component of the epidermal permeability barrier. Previous studies showed that GlcCer synthase (GCS) activity increases during <em>keratinocyte</em> differentiation; however, the mechanism by which GCS activity is regulated was not established. In the present study, we prepared anti-peptide antibodies and amplified cDNA probes based on the cDNA sequence for human GCS (Ichikawa, S., Sakiyama, H., Suzuki, G., Hidari, K. I.-P. J., and Hirabayashi, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4638-4643) in order to study GCS expression during <em>keratinocyte</em> differentiation. Confluent human <em>keratinocytes</em> in culture were induced to terminally differentiate by elevation of Ca+<em>2</em> in the medium without exogenous hormones or <em>growth</em> <em>factors</em>. GlcCer synthesis assayed in situ using a fluorescent ceramide analog increased approximately 5-fold during <em>keratinocyte</em> differentiation, peaking at day 6. Fluorescence microscopy studies of living <em>keratinocytes</em> showed that fluorescent ceramide and/or its metabolites accumulated in the Golgi in undifferentiated cells but targeted to unique vesicular structures that may be derived from the trans-Golgi region. Expression of both GCS mRNA, a approximately 3. 8-kilobase transcript on Northern blots, and GCS protein, a approximately 38-kDa polypeptide detected by Western blotting, increased dramatically (approximately 5-fold) during differentiation, reaching a maximum at about day 8. These results suggest that GCS is up-regulated at the transcriptional level during <em>keratinocyte</em> differentiation and provide the first direct evidence for GCS up-regulation in any cell type.

In the EAhy9<em>2</em>6 endothelial cell line, UTP, ATP, and forskolin, but not UDP and epidermal <em>growth</em> <em>factor</em>, inhibited tumor necrosis <em>factor</em> alpha (TNFalpha)- and sorbitol stimulation of the stress-activated protein kinases, JNK, and p38 mitogen-activated protein (MAP) kinase, and MAPKAP kinase-<em>2</em>, the downstream target of p38 MAP kinase. In NCT<em>2</em>544 <em>keratinocytes</em>, UTP and a proteinase-activated receptor-<em>2</em> agonist caused similar inhibition, but in 131<em>2</em>1N1 cells, transfected with the human P<em>2</em>Y(<em>2</em>) or P<em>2</em>Y(4) receptor, UTP stimulated JNK and p38 MAP kinase activities. This suggests that the effects mediated by P<em>2</em>Y receptors are cell-specific. The inhibitory effects of UTP were not due to induction of MAP kinase phosphatase-1, but were manifest upstream in the pathway at the level of MEK-4. The inhibitory effect of UTP was insensitive to the MEK-1 inhibitor PD 098059, changes in intracellular Ca(<em>2</em>+) levels, or pertussis toxin. Acute phorbol 1<em>2</em>-myristate 13-acetate pretreatment also inhibited TNFalpha-stimulated SAP kinase activity, while chronic pretreatment reversed the effects of UTP. Furthermore, the protein kinase C inhibitors Ro318<em>2</em><em>2</em>0 and Go6983 reversed the inhibitory action of UTP, but GF109<em>2</em>03X was ineffective. These results indicate a novel mechanism of cross-talk regulation between P<em>2</em>Y receptors and TNFalpha-stimulated SAP kinase pathways in endothelial cells, mediated by Ca(<em>2</em>+)-independent isoforms of protein kinase C.

1. The present study examined the role of C-phycocyanin (C-pc) in relation to <em>growth</em> <em>factors</em> and cell migration during wound healing. <em>2</em>. Histological and biochemical studies showed that C-pc scaffold significantly (P < 0.01) increased hydroxyl proline, total hexamine and protein content, and decreased uronic acid content in the wound tissues during a time course study in newly formed skin. 3. Reverse transcription polymerase chain reaction array of mouse <em>growth</em> <em>factors</em> in wound tissue showed overexpression (up to 10-fold) of <em>growth</em> <em>factors</em>, such as Cxcl1<em>2</em>, Fgf18, Lefty 1, Lefty <em>2</em>, Rabep 1 and Zip91, and downregulation (up to -10-fold) of Amh, Bmp 7 and Nodal genes in a 6-day period in C-pc treated groups. Also, Csf 3, Fgf <em>2</em><em>2</em>, Mdk, Igf <em>2</em>, transforming <em>growth</em> <em>factor</em> (TGF)-α 1 and interleukin (IL)-1β showed an upregulation of more than 30-fold than the control groups. TGF-β subfamily cytokine <em>growth</em> <em>factors</em>, such as Bmp <em>2</em>, 4 and 8b, and other <em>growth</em> <em>factors</em>, such as Cxcl 1, showed the highest activity on day 3, showing a transient type of regulation. Western blot analysis showed a positive correlation between gene activity and protein expressions of Bmp 8b, Bmp4, Bmp<em>2</em> and Cxcl 1. Day 6 in the C-pc group showed the highest csf3 and IL-1β expression. 4. C-pc had no direct effect on <em>keratinocyte</em> migration. However, <em>keratinocytes</em> that were co-cultured with fibroblasts showed a significantly higher rate of migration in the presence of C-pc, showing an indirect effect of C-pc on <em>keratinocyte</em> migration. 5. In conclusion, biodegradable C-pc scaffold might help to serve as an alternate scaffold material for wound healing.

We monitored cyclooxygenase-<em>2</em> (COX-<em>2</em>) expression in the insulin-like <em>growth</em> <em>factor</em>-II (IGF-II) treated human <em>keratinocytes</em> and explored the IGF-II signaling pathways with respect to the expression of COX-<em>2</em>. IGF-II induced COX-<em>2</em> mRNA and protein levels, and the up-regulation of COX-<em>2</em> expression by IGF-II was reduced by pretreatment with inhibitors of tyrosine kinase, Src and PI3-kinase. The inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) 1 also reduced the increased expression of COX-<em>2</em> by IGF-II, but the inhibition of p38 did not. To further examine the roles of these mitogen-activated protein kinases (MAPKs) in IGF-II-induced COX-<em>2</em> expression, we performed COX-<em>2</em> promoter analysis using dominant negative plasmids of MEK1 (DN-MEK1), p38 (DN-p38) and JNK1 (DN-JNK1). Although IGF-II increased COX-<em>2</em> promoter activity approximately <em>2</em>.5-fold, this increase was blocked by cotransfection with DN-MEK1 or DN-JNK1. However, DN-p38 did not block the IGF-II-induced COX-<em>2</em> promoter activity. In addition, inhibition of ERK or JNK1 reduced the increase of IGF-II-induced prostaglandin E(<em>2</em>) synthesis or cell proliferation. These results suggest that IGF-II induces COX-<em>2</em> expression through the tyrosine kinase-Src-ERK and tyrosine kinase-PI3-kinase pathways, but not via p38 MAPK pathway, and that the basal JNK activity is required for the upregulation of COX-<em>2</em> by IGF-II, as well.

In response to cutaneous injury, expression of collagenase-1 is induced in <em>keratinocytes</em> via alpha<em>2</em>beta1 contact with native type I collagen, and enzyme activity is essential for cell migration over this substratum. However, the cellular mechanism(s) mediating integrin signaling remain poorly understood. We demonstrate here that treatment of <em>keratinocytes</em> cultured on type I collagen with epidermal <em>growth</em> <em>factor</em> receptor (EGFR) blocking antibodies or a specific receptor antagonist inhibited cell migration across type I collagen and the matrix-directed stimulation of collagenase-1 production. Additionally, stimulation of collagenase-1 expression by hepatocyte <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-beta1, and interferon-gamma was blocked by EGFR inhibitors, suggesting a required EGFR autocrine signaling step for enzyme expression. Collagenase-1 mRNA was not detectable in <em>keratinocytes</em> isolated immediately from normal skin, but increased progressively following <em>2</em> h of contact with collagen. In contrast, EGFR mRNA was expressed at high steady-state levels in <em>keratinocytes</em> isolated immediately from intact skin but was absent following <em>2</em> h cell contact with collagen, suggesting down-regulation following receptor activation. Indeed, tyrosine phosphorylation of the EGFR was evident as early as 10 min following cell contact with collagen. Treatment of <em>keratinocytes</em> cultured on collagen with EGFR antagonist or heparin-binding (HB)-EGF neutralizing antibodies dramatically inhibited the sustained expression (6-<em>2</em>4 h) of collagenase-1 mRNA, whereas initial induction by collagen alone (<em>2</em> h) was unaffected. Finally, expression of collagenase-1 in ex vivo wounded skin and re-epithelialization of partial thickness porcine burn wounds was blocked following treatment with EGFR inhibitors. These results demonstrate that <em>keratinocyte</em> contact with type I collagen is sufficient to induce collagenase-1 expression, whereas sustained enzyme production requires autocrine EGFR activation by HB-EGF as an obligatory intermediate step, thereby maintaining collagenase-1-dependent migration during the re-epithelialization of epidermal wounds.

Inhibitor of <em>growth</em>-4 (ING4) is a member of the ING family and acts as a tumor suppressor protein. ING4 is a promising candidate for cancer research due to its anti-angiogenic function and its role in the inhibition of cell migration, cell cycle, and induction of apoptosis. Interaction of this protein with the histone acetyl transferase complex plays a vital role in the regulation of multiple nuclear <em>factor</em> kappa light chain enhancer of activated B cells response elements and thus in the regulation of innate immunity. Splice variants of ING4 have different binding affinities to target sites, which results in the enhancement of its functional diversity. ING4 is among the few known regulatory proteins that can directly interact with chromatin as well as with transcription <em>factors</em>. The influence of ING4 on tumor necrosis <em>factor</em>-α, <em>keratinocyte</em> chemoattractant, interleukin (IL)-6, IL-8, matrix metalloproteinases, cyclooxygenase-<em>2</em>, and IκBα expression clearly demonstrates its critical role in the regulation of inflammatory mediators. Its interaction with liprin α1 and p53 contribute to mitigate cell spreading and induce apoptosis of cancer cells. Multiple <em>factors</em> including breast cancer melanoma suppressor-1 are upstream regulators of ING4 and are frequently deactivated in tumor cells. In the present review, the different properties of ING4 are discussed, and its activities are correlated with different aspects of cell physiology. Special emphasis is placed on our current understanding of ING4 with respect to its influence on chromatin modification, tumorigenesis, and innate immunity.

Reepithelialization of human suction blister wounds was examined in five normal human volunteers over a period of 14 days postwounding to understand the control of <em>keratinocyte</em> migration, proliferation, and differentiation in acute wound healing in a controlled model. The hypothesis that morphological changes and progenitor activation result from altered cytokines and <em>growth</em> <em>factor</em> expression [in particular interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), transforming <em>growth</em> <em>factor</em> alpha (TGF-alpha), TGF-beta 1, and <em>keratinocyte</em> <em>growth</em> <em>factor</em>] was tested using semiquantitative immunohistochemistry combined with reverse transcriptase-polymerase chain reaction of samples from the blister roof, edge, and base. Parallel changes in keratin expression were examined using a wide range of well-established antibodies to multiple keratins and in situ hybridization for keratin 16 (K16), a marker of the hyperproliferative (mucoregenerative) phenotype. Longitudinal morphological, semiquantitative cytokine and <em>growth</em> <em>factor</em> expression, and histometric histone and cytokeratin profiles suggest three phases to reepithelialization: phase 1, or the acute activation phase, early in the first <em>2</em>4 hours postwounding is characterized by epidermal expression of IL-1beta and IL-6, and dermal expression of TGF-beta1, as basal, upper outer root sheath, and putative interfollicular transit amplifying <em>keratinocytes</em> become committed to mitosis; phase <em>2</em>, or the early activation phase, late in the second <em>2</em>4 hours postwounding, characterized by epidermal expression of TGF-alpha and IL-6 with concurrent suprabasal K16 expression and migration with continued proliferation, and dermal expression of <em>keratinocyte</em> <em>growth</em> <em>factor</em> and IL-6; and phase 3 or restitution over the following <em>2</em> weeks, characterized by the return of normal homeostasis, including bulge activation as evidenced by K19 expression.

A membrane-surface glycoprotein, RANDAM-<em>2</em>, is one of the neuronal cell lineage-specific antigens involved in the neuronal differentiation of P19 embryonic carcinoma (EC) cells and the mouse central nervous system (CNS). Complementary DNA cloning of RANDAM-<em>2</em> indicated that its nucleotide sequence completely matched that of PA<em>2</em>.<em>2</em>6 antigen, a sialomucin-like transmembrane glycoprotein previously found on tumorigenic <em>keratinocytes</em>. RANDAM-<em>2</em> transcripts were detectable from the embryonic stage of 6.5 days, and then the expression continued throughout the remaining embryonic stages and adulthood, with a localization restricted to the CNS. In <em>growth</em> <em>factor</em>-induced neurospheres and adult cerebrum, RANDAM-<em>2</em>-expressing cells coincided well not only with nestin-positive cells but also with glutamate-positive neurons, but not with gamma-aminobutyric acid-positive ones. These results indicate that RANDAM-<em>2</em> is one of the type I membrane surface antigens constitutively expressed on undifferentiated neuronal cells and the glutamatergic neuronal cells during mouse neurogenesis.

Breast cancer metastasis is directly associated with breast cancer cell motility. Using a cell culture wounding model, we have demonstrated that <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) enhanced the motility of estrogen receptor-positive breast cancer cells. However, the mechanisms by which KGF enhanced motility of breast cancer cells are not known. In the present study, we report that KGF-induced motility requires intact tyrosine kinase signaling since genistein, a tyrosine kinase inhibitor, led to decreased motility of breast cancer cells mediated by KGF. Using cDNA microarrays, we previously found that KGF increased the expression of Grb<em>2</em> mRNA by <em>2</em> 3-fold. Since Grb<em>2</em> plays an important role in tyrosine kinase signaling, we examined the involvement of Grb<em>2</em> in KGF-induced motility. Down-regulation of Grb<em>2</em> protein expression inhibited KGF-induced motility. Since Grb<em>2</em> is known to regulate Erk1,<em>2</em> and Akt kinase activities we determined whether these downstream proteins may be vital to KGF-induced motility. Inhibiting the activation of Erk1,<em>2</em> by PD98059 suppressed KGF-induced motility whereas inhibiting the activation of Akt by wortmannin did not affect KGF-induced motility. In conclusion, these results indicate that KGF mediated signal transduction employs Grb<em>2</em> to transduce the tyrosine kinase signals resulting in the activation of Erk1,<em>2</em> and breast cancer cell motility.

In this study we have investigated epidermal <em>growth</em> and differentiation during wound healing in human skin. The studies were performed in excisional wounds in normal skin and in chronic venous ulcers. Tissues were analyzed by immunohistochemical staining for proliferation-associated nuclear antigens (PCNA and Ki-67 antigen) and cytokeratin 16. Healing of excisional wounds was studied from day <em>2</em> to 14. Recruitment of resting (G0) epidermal cells started within <em>2</em> days after wounding; the number of cycling cells was maximal at day 4 and continued to be increased (compared to baseline levels in normal skin) after wound closure (7-14 days). Cytokeratin 16, a proliferation-associated keratin, was induced within 48 h and was expressed in the suprabasal <em>keratinocytes</em> of the wound edge. Cytokeratin 16 expression was maximal at day 4 and was still present in the neo-epidermis after restoration of epidermal continuity (7-14 days). Surprisingly, in chronic venous ulcers, cycling cells were present in the wound edges of all stages of the leg ulcers studied. Both the number and localization of cycling cells were similar to those in normal wound healing. Cytokeratin 16 was strongly expressed in all these ulcers. Our in vivo data demonstrate that recruitment of G0-cells into the cell cycle is not impaired in venous ulcers, which suggests that epidermal proliferation is not a limiting <em>factor</em> in the healing process of chronic venous ulcers.

Synapsins are pre-synaptic vesicle-associated proteins linked to the pathogenesis of epilepsy through genetic association studies in humans. Deletion of synapsins causes an excitatory/inhibitory imbalance, exemplified by the epileptic phenotype of synapsin knockout mice. These mice develop handling-induced tonic-clonic seizures starting at the age of about 3 months. Hence, they provide an opportunity to study epileptogenic alterations in a temporally controlled manner. Here, we evaluated brain inflammation, synaptic protein expression, and adult hippocampal neurogenesis in the epileptogenic (1 and <em>2</em> months of age) and tonic-clonic (3.5-4 months) phase of synapsin <em>2</em> knockout mice using immunohistochemical and biochemical assays. In the epileptogenic phase, region-specific microglial activation was evident, accompanied by an increase in the chemokine receptor CX3CR1, interleukin-6, and tumor necrosis <em>factor</em>-α, and a decrease in chemokine <em>keratinocyte</em> chemoattractant/ <em>growth</em>-related oncogene. Both post-synaptic density-95 and gephyrin, scaffolding proteins at excitatory and inhibitory synapses, respectively, showed a significant up-regulation primarily in the cortex. Furthermore, we observed an increase in the inhibitory adhesion molecules neuroligin-<em>2</em> and neurofascin and potassium chloride co-transporter KCC<em>2</em>. Decreased expression of γ-aminobutyric acid receptor-δ subunit and cholecystokinin was also evident. Surprisingly, hippocampal neurogenesis was reduced in the epileptogenic phase. Taken together, we report molecular alterations in brain inflammation and excitatory/inhibitory balance that could serve as potential targets for therapeutics and diagnostic biomarkers. In addition, the regional differences in brain inflammation and synaptic protein expression indicate an epileptogenic zone from where the generalized seizures in synapsin <em>2</em> knockout mice may be initiated or spread.

Arsenic (As) is both a human carcinogen and an effective anticancer drug. These aspects of arsenic toxicity develop as a consequence of arsenic-induced oxidative stress and modifications to signal pathway activity which alter gene expression. Resveratrol (RVL) a food antioxidant found in grapes and other fruits, exhibits anti-carcinogenic properties by reducing oxidative stress and restoring signal pathway control. This study investigated the impact of RVL on arsenite [As(III)]-induced cell signalling in HaCaT <em>keratinocytes</em> by assaying phosphorylation status of epidermal <em>growth</em> <em>factor</em> receptor (EGFR) signalling intermediates and measuring changes in expression of Phase II and DNA repair biomarkers. As(III) exposure produced dose-dependent toxicity which was associated with increased activation of EGFR pathway intermediates, cSrc, Rac1 and extracellular signal-regulated kinases 1 and <em>2</em> (ERK1/<em>2</em>). Arsenic-mediated ERK1/<em>2</em> activation negatively regulated DNA polymerase beta expression and up regulated heme-oxygenase-1 at toxic concentrations. RVL treatment modulated As(III)-mediated ERK1/<em>2</em> activation by shifting the balance of cSrc regulatory domain phosphorylation. These effects significantly altered the response of the EGFR pathway to <em>growth</em> <em>factor</em>-induced stimulation. Our research provides evidence that treatment with pharmacologically relevant doses of RVL influences cellular responses to As(III), largely due to RVL-mediated changes to Src and ERK1/<em>2</em> activation.

OBJECTIVE

Inside-out signaling occurs when changes in organellar activity lead to alterations in cell signaling that culminate at the cell surface. Mitochondria are vital signaling platforms in cells that participate in radiation-induced inside-out signaling. However, the importance of the reactive oxygen species (ROS)-scavenging ability of mitochondria through manganese superoxide dismutase (MnSOD) is not established. Here, we used MnSOD heterozygous knockout and transgenic SKH-1 hairless, albino mice and MnSOD knockdown and overexpressing HaCaT human keratinocytes to study the effects of MnSOD on ultraviolet (UV) radiation-induced inside-out signaling.

UNASSIGNED

There is an inverse correlation between MnSOD expression and UV-induced activation of epidermal growth factor receptor (EGFR), as determined by phosphorylation at Tyr1068, both in vitro and in vivo, which correlates with increased ROS production (as measured by dihydroethidium fluorescence). EGFR activation is dependent on Nox4 expression and Src kinase activation, with Src activation upstream of Nox4 in regulation of EGFR activation. Enhanced EGFR activation in MnSOD knockdown cells is abrogated by treatment with the SOD mimetic MnTnBuOE-2-PyP(5+).

CONCLUSIONS

Our data demonstrate that the ROS-scavenging ability of mitochondria, through the expression of MnSOD, is important for UV-induced inside-out signaling. Decreased MnSOD expression enhances UV-induced activation of different oncogenic signaling pathways through an inside-out signaling-mediated mechanism. Inhibition of inside-out signaling by MnTnBuOE-2-PyP(5+) mimics the effect of endogenous MnSOD, suggesting that pharmacological intervention by SOD mimetics could play an important role in the prevention of aberrant cell signaling, which may contribute to carcinogenesis and may prove valuable for the treatment or prevention of cancer in the future.

Because most human squamous carcinoma cell lines of the aerodigestive and genital tracts are refractory to the antiproliferative action of transforming <em>growth</em> <em>factor</em> beta 1 (TGF beta 1) in vitro, we have begun to identify the causes for resistance of squamous carcinoma cell lines to TGF beta 1 by using somatic cell genetics. Two stable hybrid cell lines (FaDu-HKc.1 and FaDu-HKc.<em>2</em>) were obtained by fusing a TGF beta 1-resistant human squamous carcinoma cell line, FaDu-HygR, with a human papilloma virus 16-immortalized, TGF beta 1-sensitive, human foreskin <em>keratinocyte</em> cell line, HKc-neoR. Whereas TGF beta 1 did not inhibit DNA synthesis in parental FaDu-HygR cells, it reduced DNA synthetic activity of HKc-neoR, FaDu-HKc.1, and FaDu-HKc.<em>2</em> cells by 75-85% (IC50, <em>2</em>-5 pM). Although squamous carcinoma cells express lower than normal levels of TGF beta 1 type II receptors on their cell surface, TGF beta 1 type II receptor mRNA was detected in all four cell lines. Recessive genes involved in TGF beta 1 signaling may be localized to the distal portion of chromosome 18q, as this was the sole chromosomal region of homozygous deletion in parental FaDu-HygR cells. Furthermore, our previous observation that mutant p53 decreases sensitivity of <em>keratinocytes</em> to TGF beta 1 was supported by the finding that the level of the mutant p53 protein expressed by the hybrid cell lines was greatly reduced. In summary, TGF beta 1 resistance of FaDu cells appears to be recessive and is presumably due to the loss of one or more post-receptor elements of the signaling pathway.

The importance of cancer-mesenchymal interactions in the aggressive behavior of scirrhous gastric cancer is supported by experimental and clinical evidences. We have previously reported that gastric fibroblasts secretion of <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) underline the remarkable proliferation of scirrhous gastric cancer cells. Cyclooxygenase-<em>2</em> (COX-<em>2</em>) is not only expressed in cancer cells, but also in interstitial fibroblasts in gastric carcinoma. To clarify the mechanisms responsible for the antiproliferation effect of COX-<em>2</em> inhibitors, effect of COX-<em>2</em> inhibitor on the paracrine epithelial-mesenchymal interactions of <em>growth</em> was examined. Scirrhous gastric cancer cell line, OCUM-<em>2</em>M, gastric fibroblasts, NF-<em>2</em>1, and COX-<em>2</em> inhibitor, JTE-5<em>2</em><em>2</em>, were used. <em>Growth</em>-interaction was examined by calculating the number of cancer cells or by measuring [(3)H] thymidine incorporation of cancer cells. Effect of JTE-5<em>2</em><em>2</em> on KGF expression from NF-<em>2</em>1 cells and OCUM-<em>2</em>M cells was analyzed by ELISA and RT-PCR. The conditioned medium from gastric fibroblasts significantly stimulated the <em>growth</em> of scirrhous gastric cancer cells. JTE-5<em>2</em><em>2</em> at the concentrations of 10(-5) and 10(-6) M significantly decreased the <em>growth</em>-stimulating activity of gastric fibroblasts. JTE-5<em>2</em><em>2</em> reduced the expression of KGF mRNA and the production of KGF from gastric fibroblasts. Oral administration of JTE-5<em>2</em><em>2</em> significantly decreased the size of xenografted tumor coinoculated with OCUM-<em>2</em>M cells and NF-<em>2</em>1 cells in nude mice. JTE-5<em>2</em><em>2</em> decreased COX-<em>2</em> expression and Ki67 labeling index within the coinoculated tumor. These findings suggested that a selective COX-<em>2</em> inhibitor, JTE-5<em>2</em><em>2</em>, downregulates KGF production from gastric fibroblasts, resulting in the inhibition of paracrine epithelial-mesenchymal interactions of proliferation between scirrhous gastric cancer cells and gastric fibroblasts.

The parathyroid hormone-like peptide (PLP) gene is widely expressed in normal and neoplastic tissues. Previous studies have demonstrated that PLP gene expression is regulated by serum and cycloheximide, features common to the regulation of a number of different early response genes. We now report that PLP mRNA transcripts are induced within 5 min of exposure of rat <em>keratinocytes</em> to serum, return to control values at <em>2</em>0 min, and then increase and remain elevated for at least 4 h, following which they return to baseline levels. The PLP mRNA t1/<em>2</em> was approximately 90 min in both serum-deprived and serum-stimulated cells. The serum induction was blocked by actinomycin D. Cycloheximide alone induced PLP gene expression; however, PLP mRNA transcripts were not superinduced in the presence of both serum and cycloheximide. Dexamethasone and 1,<em>2</em>5-dihydroxyvitamin D3 inhibited the basal levels of PLP mRNA transcripts but did not eliminate the serum induction of PLP gene expression. Epidermal <em>growth</em> <em>factor</em> or transforming <em>growth</em> <em>factor</em>-beta alone induced PLP mRNA transcripts, but no induction was observed following exposure of cells to epidermal <em>growth</em> <em>factor</em> and transforming <em>growth</em> <em>factor</em>-beta together. Treatment with 1<em>2</em>-O-tetradecanoylphorbol-13-acetate for 90 min did not induce PLP mRNA transcripts, but 1<em>2</em>-O-tetradecanoylphorbol-13-acetate blocked the rapid serum induction of PLP gene expression. These features of PLP gene expression suggest that PLP is a member of the <em>growth</em> <em>factor</em>-regulated early response gene family. The rapid serum stimulation of PLP gene expression raises the possibility that PLP may contribute in an autocrine fashion to the early cellular response to <em>growth</em> <em>factor</em> stimulation.

Parathyroid hormone-related protein (PTHrP) is produced by a wide range of neoplastic and normal cells, including <em>keratinocytes</em> where it may regulate <em>growth</em> and differentiation. Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) is a <em>growth</em> <em>factor</em> produced by many cells, including <em>keratinocytes</em> where it regulates epidermal homeostasis. TGF-beta has been reported to be cosecreted with PTHrP in some neoplasms and to stimulate PTHrP production by neoplastic <em>keratinocytes</em>. However, the effects of TGF-beta on PTHrP production by normal <em>keratinocytes</em> are not well characterized. In this study, we investigated the effects of endogenous and exogenous TGF-beta on PTHrP production by normal human foreskin <em>keratinocytes</em>. PTHrP secretion, mRNA expression, and mRNA transcription in vitro were determined by N-terminal radioimmunoassay, ribonuclease protection assay, and transient transfections. PTHrP production and secretion of latent TGF-beta activity were greatest in proliferating <em>keratinocytes</em> prior to and at confluence of monolayer cultures. TGF-beta1 increased PTHrP mRNA expression by normal <em>keratinocytes</em> in a dose-dependent manner with maximal stimulation at 6-1 <em>2</em> h after treatment. In addition, <em>keratinocytes</em> treated with a monoclonal anti-TGF-beta antibody expressed decreased levels of PTHrP mRNA. The increased levels of PTHrP mRNA following TGF-beta1 treatment were owing, at least partly, to an increase in PTHrP mRNA stability. TGF-beta1 failed to activate transcription of the luciferase reporter gene driven by either the human or mouse PTHrP promoters. In conclusion, TGF-beta1 functions as a paracrine or autocrine regulator of PTHrP production in normal human <em>keratinocytes</em>, and this may play a role in the regulation of <em>keratinocyte</em> proliferation or differentiation.

It has been demonstrated that hyperthermia protects <em>keratinocytes</em> from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if <em>keratinocytes</em> become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-delta(1<em>2</em>,14)-prostaglandin J<em>2</em> (15dPGJ<em>2</em>) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal <em>keratinocytes</em> (NHEK) and the carcinoma-derived cell line A431 were exposed to either 4<em>2</em> degrees C or to 15dPGJ<em>2</em> for 4 hours at <em>2</em>4-hour intervals for 4 consecutive days. The intracellular level of the 7<em>2</em>-kDa heat shock protein (Hsp7<em>2</em>) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ<em>2</em> treatment, and survival was determined <em>2</em>4 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ<em>2</em> are potent inducers of Hsp7<em>2</em> expression and lead to increased resistance to UVB-induced cell death in human <em>keratinocytes</em>; (<em>2</em>) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp7<em>2</em> and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ<em>2</em> is enhanced by repeated exposure without a further increase of Hsp7<em>2</em>; (4) repeated heat shock and 15dPGJ<em>2</em> up to a concentration of 1 microg/mL have no influence on cell <em>growth</em> over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing <em>factors</em>, stress tolerance can be maintained without additional toxicity in human <em>keratinocytes</em>. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.

In the present study we prepared explant cultures of plucked total hair follicles and of fragments microdissected from the following regions: B1 (bulb region), B<em>2</em> (intermediate region), B3-1 (lower central outer root sheath, ORS), B3-<em>2</em> (upper central ORS) and B4 (area of fracture). The <em>growth</em> capacities, the start of epithelial out<em>growth</em>, the stages of differentiation and apoptosis were studied immunohistochemically in early and late explant cultures using a battery of antibodies against cytokeratins, <em>growth</em> <em>factor</em> receptors and cell adhesion molecules and proliferation markers. Whole plucked hair follicles showed epithelial out<em>growth</em>s exclusively in the upper central ORS (B3-<em>2</em>) starting early, mostly by day 3. In microdissected fragments, in contrast, out<em>growth</em>s were more widespread, mostly in B3-<em>2</em> and B3-1, and started early, but were also of late onset in some cases of B<em>2</em> and B4. Epithelial out<em>growth</em>s exhibited a basal layer of small cuboidal cells in a low stage of differentiation and one to two suprabasal layers of large prickle-like cells expressing late differentiation markers. The former expressed the receptor of nerve <em>growth</em> <em>factor</em> (NGF) heterogeneously whereas epidermal <em>growth</em> <em>factor</em> (EGF) receptor was not detectable. This is similar to ORS cells of this area in vivo. The proliferative activity of the out<em>growth</em>s was always restricted to peripheral cells. Thus no essential differences in differentiation of outgrowing cells were detected. These results suggest that <em>keratinocytes</em> with the highest <em>growth</em> capacities in plucked human hair follicles are localized in the lower central ORS (corresponding to B3-<em>2</em>) and some with a lower capacity in the upper central ORS (corresponding to B3-1) as established after microdissection. This is in agreement with the bulge activation theory. NGF may also play a role in hair <em>growth</em>.

BACKGROUND

Malaria is the most relevant parasitic disease worldwide, and still accounts for 1 million deaths each year. Since current antimalarial drugs are unable to prevent death in severe cases, new therapeutic strategies have been developed. Mesenchymal stromal cells (MSC) confer host resistance against malaria; however, thus far, no study has evaluated the therapeutic effects of MSC therapy on brain and distal organ damage in experimental cerebral malaria.

METHODS

Forty C57BL/6 mice were injected intraperitoneally with 5 × 10(6) Plasmodium berghei-infected erythrocytes or saline. After <em>2</em>4 h, mice received saline or bone marrow (BM)-derived MSC (1x10(5)) intravenously and were housed individually in metabolic cages. After 4 days, lung and kidney morphofunction; cerebrum, spleen, and liver histology; and markers associated with inflammation, fibrogenesis, and epithelial and endothelial cell damage in lung tissue were analyzed.

RESULTS

In P. berghei-infected mice, BM-MSCs: 1) reduced parasitemia and mortality; <em>2</em>) increased phagocytic neutrophil content in brain, even though BM-MSCs did not affect the inflammatory process; 3) decreased malaria pigment detection in spleen, liver, and kidney; 4) reduced hepatocyte derangement, with an increased number of Kupffer cells; 5) decreased kidney damage, without effecting significant changes in serum creatinine levels or urinary flow; and 6) reduced neutrophil infiltration, interstitial edema, number of myofibroblasts within interstitial tissue, and collagen deposition in lungs, resulting in decreased lung static elastance. These morphological and functional changes were not associated with changes in levels of tumor necrosis <em>factor</em>-α, <em>keratinocyte</em>-derived chemokine (KC, a mouse analog of interleukin-8), or interferon-γ, which remained increased and similar to those of P. berghei animals treated with saline. BM-MSCs increased hepatocyte <em>growth</em> <em>factor</em> but decreased VEGF in the P. berghei group.

CONCLUSIONS

BM-MSC treatment increased survival and reduced parasitemia and malaria pigment accumulation in spleen, liver, kidney, and lung, but not in brain. The two main organs associated with worse prognosis in malaria, lung and kidney, sustained less histological damage after BM-MSC therapy, with a more pronounced improvement in lung function.

The in vivo cellular microenvironment is regulated by a complex interplay of soluble <em>factors</em> and signaling molecules secreted by cells and it plays a critical role in the <em>growth</em> and development of normal and diseased tissues. In vitro systems that can recapitulate the microenvironment at the cellular level are needed to investigate the influence of autocrine signaling and extracellular matrix effects on tissue homeostasis, regeneration, disease development and progression. In this study, we report the use of microbubble technology as a means to culture cells in a controlled microenvironment in which cells can influence their function through autocrine signaling. Microbubbles (MB) are small spherical cavities about 100-300 μm in diameter formed in hydrophobic polydimethylsiloxane (PDMS) with ∼60-100 μm circular openings and aspect ratio ∼3.0. We demonstrate that the unique architecture of the microbubble compartment is advantaged for cell culture using HaCaT cells, an immortalized <em>keratinocyte</em> cell line. We observe that HaCaT cells, seeded in microbubbles (15-<em>2</em>0 cells/MB) and cultured under standard conditions, adopt a compact 3D spheroidal morphology. Within <em>2</em>-3 days, the cells transition to a sheeting morphology. Through experimentation and simulation we show that this transition in morphology is due to the unique architecture of the microbubble compartment which enables cells to condition their local microenvironment. The small media volume per cell and the development of shallow concentration gradients allow <em>factors</em> secreted by the cells to rise to bioactive levels. The kinetics of the morphology transition depends on the number of cells seeded per microbubble; higher cell seeding induces a more rapid transition. HaCaT cells seeded onto PDMS cured in 96-well plates also form compact spheroids but they do not undergo a transition to a sheeting morphology even after several weeks of culture. The importance of soluble <em>factor</em> accumulation in driving this morphology transition in microbubbles is supported by the observation that spheroids do not form when cells - seeded into microbubbles or onto PDMS cured in 96-well plates - are cultured in media conditioned by HaCaT cells grown in standard tissue culture plate. We observed that the addition of TGF-β1 to the <em>growth</em> media induced cells to proliferate in a sheeting morphology from the onset both on PDMS cured in 96-well plates and in microbubbles. TGF-β1 is a morphogen known to regulate epithelial-to-mesenchymal transition (EMT). Studies of the role of Ca(<em>2</em>+) concentration and changes in E-cadherin expression additionally support an EMT-like HaCaT morphology transition. These findings taken together validate the microbubble compartment as a unique cell culture platform that can potentially transform investigative studies in cell biology and in particular the tumor microenvironment. Targeting the tumor microenvironment is an emerging area of anti-cancer therapy.