<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), also termed as fibroblast <em>growth</em> <em>factor</em>-7, promotes proliferation, migration, and adhesion of skin <em>keratinocytes</em> via binding to <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor (KGFR) and subsequent activation of downstream signaling including the PI3K-AKT-mTORC1 pathway. Here, we found that the α-subunits of the G proteins (Gαi1/3) and <em>growth</em> <em>factor</em> receptor binding <em>2</em>-associated binding protein 1 (Gab1) are required for this activation process. With KGF stimulation, Gαi1/3 formed a complex with KGFR and was required for subsequent Gab1 recruitment, phosphorylation, and following PI3K-p85 activation. In addition, Gαi1/3 short hairpin RNA knockdown largely inhibited KGF-induced cell proliferation, migration, and the accumulation of cyclin D1/fibronectin in cultured skin <em>keratinocytes</em>. Furthermore, we observed increased expression of Gαi1/3 in wounded human skin and keloid skin tissues, suggesting the possible involvement of Gαi1/3 in wound healing and keloid formation. Overall, we suggest that Gαi1/3 proteins lie downstream of KGFR, but upstream of Gab1-mediated activation of PI3K-AKT-mTORC1 signaling, thus revealing a role for Gαi proteins in mediating KGFR signaling, cell migration, and possible wound healing.

Staphylococcal alpha-toxin is an archetypal killer protein that homo-oligomerizes in target cells to create small transmembrane pores. The membrane-perforating beta-barrel motif is a conserved attack element of cytolysins of Gram-positive and Gram-negative bacteria. Following the recognition that nucleated cells can survive membrane permeabilization, a profile of abundant transcripts was obtained in transiently perforated <em>keratinocytes</em>. Several immediate early genes were found to be upregulated, reminiscent of the cellular response to <em>growth</em> <em>factors</em>. Cell cycle analyses revealed doubling of S + G<em>2</em>/M phase cells <em>2</em>6 h post toxin treatment. Determination of cell counts uncovered that after an initial drop, numbers increased to exceed the controls after <em>2</em> days. A non-lytic alpha-toxin mutant remained without effect. The alpha-toxin pore is too small to allow egress of cytosolic <em>growth</em> <em>factors</em>, and evidence was instead obtained for <em>growth</em> signalling via the epidermal <em>growth</em> <em>factor</em> receptor (EGFR). Inhibition of the EGFR or of EGFR-proligand-processing blocked the mitogenic effect of alpha-toxin. Western blots with phospho-specific antibodies revealed activation of the EGFR, and of the adapter protein Shc. Immediate early response and proliferation upon transient plasma membrane pore formation by bacterial toxins may represent a novel facet of the complex interaction between pathogen and host.

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication of malaria, and its pathophysiology is insufficiently understood. Both in humans and in murine models, MA-ARDS is characterized by marked pulmonary inflammation. We investigated the role of hemozoin in MA-ARDS in C57Bl/6 mice infected with Plasmodium berghei NK65, P. berghei ANKA, and P. chabaudi AS. By quantifying hemozoin in the lungs and measuring the disease parameters of MA-ARDS, we demonstrated a highly significant correlation between pulmonary hemozoin concentrations, lung weights, and alveolar edema. Histological analysis of the lungs demonstrated that hemozoin is localized in phagocytes and infected erythrocytes, and only occasionally in granulocytes. Species-specific differences in hemozoin production, as measured among individual schizonts, were associated with variations in pulmonary pathogenicity. Furthermore, both pulmonary hemozoin and lung pathology were correlated with the number of infiltrating inflammatory cells, an increased pulmonary expression of cytokines, chemokines, and enzymes, and concentrations of alveolar vascular endothelial <em>growth</em> <em>factor</em>. The causal relationship between hemozoin and inflammation was investigated by injecting P. falciparum-derived hemozoin intravenously into malaria-free mice. Hemozoin potently induced the pulmonary expression of proinflammatory chemokines (interferon-γ inducible protein-10/CXC-chemokine ligand (CXCL)10, monocyte chemotactic protein-1/CC-chemokine ligand <em>2</em>, and <em>keratinocyte</em>-derived chemokine/CXCL1), cytokines (IL-1β, IL-6, IL-10, TNF, and transforming <em>growth</em> <em>factor</em>-β), and other inflammatory mediators (inducible nitric oxide synthase, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate- oxidase-<em>2</em>, and intercellular adhesion molecule-1). Thus, hemozoin correlates with MA-ARDS and induces pulmonary inflammation.

Activating transcription <em>factor</em> <em>2</em> (ATF<em>2</em>) regulates transcription in response to stress and <em>growth</em> <em>factor</em> stimuli. Here, we use a mouse model in which ATF<em>2</em> was selectively deleted in <em>keratinocytes</em>. Crossing the conditionally expressed ATF<em>2</em> mutant with K14-Cre mice (K14.ATF<em>2</em>(f/f)) resulted in selective expression of mutant ATF<em>2</em> within the basal layer of the epidermis. When subjected to a two-stage skin carcinogenesis protocol [7,1<em>2</em>-dimethylbenz[a]anthracene/phorbol 1<em>2</em>-tetradecanoate 13-acetate (DMBA/TPA)], K14.ATF<em>2</em>(f/f) mice showed significant increases in both the incidence and prevalence of papilloma development compared with the WT ATF<em>2</em> mice. Consistent with these findings, <em>keratinocytes</em> of K14.ATF<em>2</em>(f/f) mice exhibit greater anchorage-independent <em>growth</em> compared with ATF<em>2</em> WT <em>keratinocytes</em>. Papillomas of K14.ATF<em>2</em>(f/f) mice exhibit reduced expression of presenilin1, which is associated with enhanced beta-catenin and cyclin D1, and reduced Notch1 expression. Significantly, a reduction of nuclear ATF<em>2</em> and increased beta-catenin expression were seen in samples of squamous and basal cell carcinoma, as opposed to normal skin. Our data reveal that loss of ATF<em>2</em> transcriptional activity serves to promote skin tumor formation, thereby indicating a suppressor activity of ATF<em>2</em> in skin tumor formation.

Reduced expression of proapoptotic and terminal differentiation genes in conjunction with increased levels of the proinflammatory and angiogenesis-inducing enzymes, cyclooxygenase <em>2</em> (COX-<em>2</em>) and inducible nitric oxide synthase (iNOS), correlate with malignant transformation of oral intraepithelial neoplasia (IEN). Accordingly, this study investigated the effects of a 10% (w/w) freeze-dried black raspberry gel on oral IEN histopathology, gene expression profiles, intraepithelial COX-<em>2</em> and iNOS proteins, and microvascular densities. Our laboratories have shown that freeze-dried black raspberries possess antioxidant properties and also induce <em>keratinocyte</em> apoptosis and terminal differentiation. Oral IEN tissues were hemisected to provide samples for pretreatment diagnoses and establish baseline biochemical and molecular variables. Treatment of the remaining lesional tissue (0.5 g gel applied four times daily for 6 weeks) began 1 week after the initial biopsy. RNA was isolated from snap-frozen IEN lesions for microarray analyses, followed by quantitative reverse transcription-PCR validation. Additional epithelial gene-specific quantitative reverse transcription-PCR analyses facilitated the assessment of target tissue treatment effects. Surface epithelial COX-<em>2</em> and iNOS protein levels and microvascular densities were determined by image analysis quantified immunohistochemistry. Topical berry gel application uniformly suppressed genes associated with RNA processing, <em>growth</em> <em>factor</em> recycling, and inhibition of apoptosis. Although the majority of participants showed posttreatment decreases in epithelial iNOS and COX-<em>2</em> proteins, only COX-<em>2</em> reductions were statistically significant. These data show that berry gel application modulated oral IEN gene expression profiles, ultimately reducing epithelial COX-<em>2</em> protein. In a patient subset, berry gel application also reduced vascular densities in the superficial connective tissues and induced genes associated with <em>keratinocyte</em> terminal differentiation.

Vascular endothelial <em>growth</em> <em>factor</em> (VEGF-A), a potent stimulus for angiogenesis, is up-regulated in the skin after wounding. Although studies have shown that VEGF is important for wound repair, it is unclear whether this is based solely on its ability to promote angiogenesis or if VEGF can also promote healing by acting directly on non-endothelial cell types. By immunohistochemistry and reverse transcriptase-polymerase chain reaction, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-<em>2</em>, was detected in murine <em>keratinocytes</em> during wound repair and in normal human epidermal <em>keratinocytes</em> (NHEKs). The presence of VEGF receptors on NHEKs was verified by binding studies with 1<em>2</em>5I-VEGF. In vitro, VEGF stimulated the proliferation of NHEKs, an effect that could be blocked by treatment with neutralizing VEGFR-1 antibodies. A role for VEGFR-1 in <em>keratinocytes</em> was also shown in vivo because treatment of excisional wounds with neutralizing VEGFR-1 antibodies delayed re-epithelialization. Treatment with anti-VEGFR-1 antibodies also reduced the number of proliferating <em>keratinocytes</em> at the leading edge of the wound, suggesting that VEGF sends a proliferative signal to these cells. Together, these data describe a novel role for VEGFR-1 in <em>keratinocytes</em> and suggest that VEGF may play several roles in cutaneous wound repair.

To localize epidermal <em>growth</em> <em>factor</em> (EGF) receptors in normal human epidermis and other skin structures, two different light microscopic methods were used. EGF binding [( 1<em>2</em>5I]EGF/R) to the extracellular portion of the EGF receptor was studied by incubating intact skin samples with [1<em>2</em>5I]EGF, sectioning the tissues, and performing autoradiography. Immunoreactive EGF receptor molecules (IR-EGF/R) were localized with a mono-specific anti-EGF receptor antibody using a <em>2</em>-step indirect immunocytochemical method (horseradish peroxidase) and detergent permeabilized tissues. This latter method measured the total pool of EGF receptors: occupied and/or internalized forms, precursor forms, and partially degraded forms of the EGF receptor that retain immunoreactivity. Both the [1<em>2</em>5I]EGF/R and IR-EGF/R localization studies indicated that EGF receptors were present in basal epidermal <em>keratinocytes</em>, sebocytes, outer root sheath cells in hair follicles, smooth muscle cells of arrector pili muscles, and dermal arteries. The highest levels of [1<em>2</em>5I]EGF/R and IR-EGF/R were found in the dermal ducts of eccrine sweat glands. The distribution of both [1<em>2</em>5I]EGF/R and IR-EGF/R was not consistent with the concept that EGF exclusively is involved in cellular division and proliferation in normal human epidermis and its appendages, i.e., EGF receptors were also found in tissues that do not undergo rapid proliferation. The present study indicates that EGF may have a more complex regulatory role in the skin than was previously thought.

Glucagon-like peptide-<em>2</em> (GLP-<em>2</em>) is a peptide hormone with multiple beneficial effects on the intestine, including expansion of the mucosal surface area through stimulation of crypt cell proliferation, as well as enhancement of nutrient digestion and absorption. Recent advances in clinical trials involving GLP-<em>2</em> necessitate elucidation of the exact signaling pathways by which GLP-<em>2</em> acts. In particular, the GLP-<em>2</em> receptor has been localized to several intestinal cell types that do not include the proliferating crypt cells, and the actions of GLP-<em>2</em> have thus been linked to a complex network of indirect mediators that induce diverse signaling pathways. The intestinotropic actions of GLP-<em>2</em> on the colon have been shown to be mediated through the actions of <em>keratinocyte</em> <em>growth</em> <em>factor</em> and insulin-like <em>growth</em> <em>factor</em> (IGF)-<em>2</em>, whereas small intestinal <em>growth</em> has been linked to IGF-1, IGF-<em>2</em>, and ErbB ligands, as well as the IGF-1 receptor and ErbB. The cellular source of these mediators remains unclear, but it likely includes the intestinal subepithelial myofibroblasts. Conversely, the anti-inflammatory and blood flow effects of GLP-<em>2</em> are dependent on vasoactive intestinal polypeptide released from submucosal enteric neurons and nitric oxide, respectively. Finally, recent studies have suggested that GLP-<em>2</em> not only modulates intestinal stem cell behavior but may also promote carcinogenesis in models of sporadic colon cancer. Further consideration of the molecular cross-talk and downstream signaling pathways mediating the intestinotropic effects of GLP-<em>2</em> is clearly warranted.

A novel Mr 17,000 heparin-binding protein was purified from culture medium conditioned by A431 human epidermoid carcinoma cells. This protein, designated HBp17, was found to bind the heparin-binding peptide <em>growth</em> <em>factors</em> HBGF-1 and HBGF-<em>2</em> in a noncovalent, reversible manner. In addition HBp17 was found to inhibit the biological activities of both HBGF-1 and HBGF-<em>2</em>. Both the binding and inactivation of HBGF-1 and HBGF-<em>2</em> by HBp17 were abolished by heparin. Full-length 1163-base pair HBp17 cDNA was cloned and sequenced by using the polymerase chain reaction technique. The deduced primary structure of HBp17 consisted of <em>2</em>34 amino acids including each of five partial peptide sequences obtained from proteolytic fragments of purified HBp17. The encoded protein included a 33-residue N-terminal signal sequence for secretion and a single potential N-linked glycosylation site. No homology with any known protein was found for the deduced primary structure of HBp17. The expression of HBp17 mRNA was found to occur preferentially in normal human <em>keratinocytes</em> and in squamous cell carcinomas. This pattern of HBp17 gene expression suggests that this binding protein for HBGFs 1 and <em>2</em> has a physiological role in squamous epithelia.

Epithelial-mesenchymal transition (EMT) is a developmental cellular process occurring during early embryo development, including gastrulation and neural crest cell migration. It can be broken down in distinct functional steps: (1) loss of baso-apical polarization characterized by cytoskeleton, tight junctions, and hemidesmosomes remodeling; (<em>2</em>) individualization of cells, including a decrease in cell-cell adhesion forces, (3) emergence of motility, and (4) invasive properties, including passing through the subepithelial basement membrane. These phases occur in an uninterrupted process, without requiring mitosis, in an order and with a degree of completion dictated by the microenvironment. The whole process reflects the activation of specific transcription <em>factor</em> families, called EMT transcription <em>factors</em>. Several mechanisms can combine to induce EMT. Some are reversible, involving <em>growth</em> <em>factors</em> and cytokines and/or environmental signals including extracellular matrix and local physical conditions. Others are irreversible, such as genomic alterations during carcinoma progression, along a selective and irreversible clonal drift. In carcinomas, these signals can converge to initiate a metastable phenotype. In this state, similarly to activated <em>keratinocytes</em> during re-epithelialization, cells can initiate a cohort migration and engage into a transient and reversible EMT controlled by the local environment prior to efficient intravasation and metastasis. EMT transcription <em>factors</em> also participate in cancer progression by inducing apoptosis resistance and maintaining stem-like properties exposed in tumor recurrences. These properties, very important on a clinical point of view, are not intrinsically linked to EMT, but can share common pathways.

The increasing ratio of ageing population poses new challenges to healthcare systems. The elderly frequently suffer from severe infections. Vaccination could protect them against several infectious diseases, but it can be effective only if cells that are capable of responding are still present in the repertoire. Recent vaccination strategies in the elderly might achieve low effectiveness due to age-related immune impairment. Immunosenescence affects both the innate and adaptive immunity.Beside individual variations of genetic predisposition, epigenetic changes over the full course of human life exert immunomodulating effects. Disturbances in macrophage-derived cytokine release and reduction of the natural killer cell mediated cytotoxicity lead to increased frequency of infections. Ageing dampens the ability of B cells to produce antibodies against novel antigens. Exhausted memory B lymphocyte subsets replace naïve cells. Decline of cell-mediated immunity is the consequence of multiple changes, including thymic atrophy, reduced output of new T lymphocytes, accumulation of anergic memory cells, and deficiencies in cytokines production. Persistent viral and parasitic infections contribute to the loss of immunosurveillance and premature exhaustion of T cells. Reduced telomerase activity and Toll-like receptor expression can be improved by chemotherapy. Reversion of thymic atrophy could be achieved by thymus transplantation, depletion of accumulated dysfunctional naive T cells and herpesvirus-specific exhausted memory cells. Administration of interleukin (IL)-<em>2</em>, IL-7, IL-10, <em>keratinocyte</em> <em>growth</em> <em>factor</em>, thymic stromal lymphopoietin, as well as leptin and <em>growth</em> hormone boost thymopoiesis. In animals, several strategies have been explored to produce superior vaccines. Among them, virosomal vaccines containing polypeptide antigens or DNA plasmids as well as new adjuvanted vaccine formulations elicit higher dendritic cell activity and more effective serologic than conventional vaccines responses in the elderly. Hopefully, at least some of these approaches can be translated to human medicine in a not too far future.

BACKGROUND

We investigated whether ghrelin is present in human saliva, is produced by salivary glands, and physiologic consequences of these findings.

METHODS

Expression of ghrelin and specific receptor mRNA was determined by PCR. Proteins were identified by immunoblotting and size-exclusion fast protein liquid chromatography (FPLC) with consecutive RIA. Specific RIAs were used for quantification of salivary total and bioactive ghrelin. Distribution of ghrelin was investigated by immunohistochemistry in cryosections of the salivary glands. The effect of ghrelin on incorporation of 5-bromo-<em>2</em>'-deoxyuridine as a measure of cell proliferation was investigated in primary oral <em>keratinocytes</em>.

RESULTS

Ghrelin is produced by the salivary glands. The hormone was identified in saliva and glands by immunoblotting and by FPLC fractionation of saliva. Immunohistochemistry demonstrated ghrelin distribution in the salivary glands. The receptor was also produced by the glands and by oral <em>keratinocytes</em> and was shown to be functional. Comparison of total ghrelin values for healthy individuals (body mass index, 18-<em>2</em>7 kg/m<em>2</em>) showed significantly lower concentrations in saliva than in serum (P < 0.01). The correlation between both matrices was r<em>2</em> = 0.56 (P < 0.001) with a negative correlation to body mass index (r<em>2</em> = 0.314; P < 0.01). Bioactive acylated ghrelin was also present in saliva. Incubation of <em>keratinocytes</em> with ghrelin led to significantly increased cell proliferation (P < 0.001). This effect could be completely suppressed by co-incubation with NOX-B11 (50 nmol/L), a novel specific inhibitor of acylated ghrelin.

CONCLUSIONS

Ghrelin in saliva is produced and released by salivary glands. The effect of ghrelin on oral cell proliferation adds to the pro-proliferative action of other salivary growth factors.

OBJECTIVE

The purpose of this study was to determine whether messenger RNA coding for hepatocyte <em>growth</em> <em>factor</em> (HGF), HGF receptor (MET), <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), KGF receptor, and fibroblast <em>growth</em> <em>factor</em> (FGF) receptor-<em>2</em> were produced in primary cultures of human corneal epithelial, stromal fibroblast, and endothelial cells, as well as ex vivo corneal epithelium, endothelial cells transfected with the SV40 large T antigen, and control embryonic lung fibroblasts. The effects of exogenous HGF and KGF, compared to epidermal <em>growth</em> <em>factor</em>, on the proliferation of first passage corneal cells were also examined.

METHODS

Polymerase chain reaction was used to amplify complementary DNA for each modulator from each cell type. Hot blotting was used to demonstrate the specificity of amplification products. Proliferation of first passage corneal epithelial, stromal fibroblast, and endothelial cells in response to varying concentrations of HGF, KGF, and epidermal growth factor was measured.

RESULTS

Specific amplification products for messenger RNA coding for each modulator were detected in each corneal cell type, although very low levels of HGF and KGF messenger RNA appeared to be present in corneal epithelial cells relative to stromal fibroblasts and corneal endothelial cells. Amplification products that may have been derived from alternative transcripts were detected for several of the modulators. HGF and KGF stimulated proliferation in a dose-response manner in first passage corneal epithelial and endothelial cells, but not stromal fibroblast cells.

CONCLUSIONS

Human corneal epithelial, stromal fibroblasts, and endothelial cells produce messenger RNA coding for HGF and KGF, although low levels appear to be present in the epithelial cells. All three major cell types of the cornea produce messenger RNA coding for HGF receptor, KGF receptor, and FGF receptor-<em>2</em>. The proliferation of human corneal epithelial and endothelial cells, but not stromal fibroblasts, was stimulated by exogenous HGF and KGF. HGF and KGF likely have intracrine, autocrine, and/or paracrine functions in the cornea. Exogenous HGF and KGF may be useful in corneal preservation and for regulating corneal wound healing.

The epidermal <em>growth</em> <em>factor</em> (EGF) receptor is highly expressed in HaCaT <em>keratinocytes</em> as shown by Western blotting. Stimulation of HaCaT cells with EGF, and also with the serine protease thrombin, induced DNA synthesis, measured by incorporation of 5-bromo-<em>2</em>'-deoxyuridine into the DNA of proliferating cells. Using antibodies directed against the active form of the EGF receptor, we show that in HaCaT cells EGF and thrombin triggered a rapid activation of the EGF receptor, followed by the phosphorylation and activation of the extracellular signal-regulated protein kinase (ERK). Moreover, EGF and thrombin induced a transient synthesis of the zinc finger transcriptional regulator Egr-1. Proliferation, activation of ERK, and biosynthesis of Egr-1 was completely inhibited in EGF or thrombin-treated HaCaT cells by the MAP kinase kinase inhibitor PD98059 and by AG1487, an EGF receptor-specific tyrosine kinase inhibitor. These data indicate that phosphorylation and activation of both the EGF receptor and ERK are essential for mitogenic signaling via EGF and thrombin. The synthesis of Egr-1 in HaCaT cells as a result of EGF or thrombin stimulation suggests that Egr-1 may be an important "late" part of the EGF and thrombin-initiated signaling cascades. We postulate that Egr-1 may function as a "third messenger" in <em>keratinocytes</em> connecting mitogenic stimulation with changes in gene transcription.

MMP-9, a member of the matrix metalloproteinase family that degrades collagen IV and processes chemokines and cytokines, participates in epidermal remodeling in response to stress and injury. Limited activity of MMP-9 is essential while excessive activity is deleterious to the healing process. Tumor necrosis <em>factor</em> (TNFalpha), a key mediator of cutaneous inflammation, is a powerful inducer of MMP-9. Calcitriol, the hormonally active vitamin D metabolite, and its analogs are known to attenuate epidermal inflammation. We aimed to examine the modulation of MMP-9 by calcitriol in TNFalpha-treated <em>keratinocytes</em>. The immortalized HaCaT <em>keratinocytes</em> were treated with TNFalpha in the absence of exogenous <em>growth</em> <em>factors</em> or active ingredients. MMP-9 production was quantified by gelatin zymography and real-time RT-PCR. Activation of signaling cascades was assessed by western blot analysis and DNA-binding activity of transcription <em>factors</em> was determined by EMSA. Exposure to TNFalpha markedly increased the protein and mRNA levels of MMP-9, while pretreatment with calcitriol dose dependently reduced this effect. Employing specific inhibitors we established that the induction of MMP-9 by TNFalpha was dependent on the activity of the epidermal <em>growth</em> <em>factor</em> receptor, c-Jun-N-terminal kinase (JNK), NFkappaB and extracellular signal-regulated kinase-1/<em>2</em>. The effect of calcitriol was associated with inhibition of JNK activation and reduction of DNA-binding activities of the transcription <em>factors</em> activator protein-1 (AP-1) and NFkappaB following treatment with TNFalpha. By down-regulating MMP-9 levels active vitamin D derivatives may attenuate deleterious effects due to excessive TNFalpha-induced proteolytic activity associated with cutaneous inflammation.

Keloids, partially considered as benign tumors, represent the most extreme example of cutaneous scarring that uniquely afflicts humans as a pathological response to wound healing. It is characterized by excessive deposition of collagen and other extracellular matrix components by dermal fibroblasts. Upon cutaneous injury, cocktails of chemokines, cytokines and <em>growth</em> <em>factors</em> are secreted temporally and spatially to direct appropriate responses from neutrophils, macrophages, <em>keratinocytes</em> and fibroblasts to facilitate normal wound healing. Signal transducer and activator of transcription 3 (Stat3) is an oncogene and a latent transcription <em>factor</em> activated by various cytokines and <em>growth</em> <em>factors</em>. We investigated the possible role of Stat3 in keloid scar pathogenesis by examining skin tissue and cultured fibroblasts from keloid-scarred patients. We observed enhanced expression and phosphorylation of Stat3 in keloid scar tissue, and in cultured keloid fibroblasts (KFs) in vitro. Increased activation of Janus kinase (Jak)<em>2</em>, but not Jak1, was detected in KFs, and suppression of Jak<em>2</em> by its inhibitor repressed Stat3 Y705 phosphorylation. Inhibition of Stat3 expression and phosphorylation by short interfering RNA or Cucurbitacin I resulted in the loss of collagen production, impaired proliferation and delayed cell migration in KFs. We show, for the first time, a role of Stat3 in keloid pathogenesis. Inhibitors of Stat3 may be useful therapeutic strategies for the prospective treatment of keloid scars.

BACKGROUND

Breast tumor kinase (Brk/protein tyrosine kinase 6 (PTK6)) is a nonreceptor, soluble tyrosine kinase overexpressed in the majority of breast tumors. Previous work has placed Brk downstream of epidermal growth factor receptor (ErbB) activation and upstream of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein (MAP) kinases. Herein we investigate the regulation of Brk kinase activity and cell migration in response to treatment of keratinocytes (HaCaT cells) and breast cancer cell lines (MDA-MB-231 and T47D cells) with hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), peptide ligands for Met and Ron receptors, respectively.

METHODS

In vitro kinase assays were performed to directly measure Brk kinase activity in response to MET and RON ligands. Transfection of Brk-targeted RNAi was used to knock down endogenous Brk or ERK5 in multiple cell lines. Kinase activities (downstream of MET signaling) were assayed by Western blotting using total and phospho-specific antibodies. Boyden chamber assays were used to measure cell migration in response to manipulation of Brk and downstream MET effectors. Rescue experiments were performed by knock down of endogenous Brk using RNAi (targeting the untranslated region (3'-UTR)) and transient transfection (re-expression) of either wild-type or kinase-inactive Brk.

RESULTS

Brk gene silencing revealed that HGF, but not MSP, induced robust Brk-dependent cell migration. Brk and ERK5 copurified in HGF-induced protein complexes, and Brk/ERK5 complexes formed independently of Brk kinase activity. ERK5 was required for breast cancer cell but not keratinocyte cell migration, which became ERK1/2-dependent upon ERK5 knockdown. Notably, rescue experiments indicated that the kinase activity of Brk was not required for HGF-induced cell migration. Further, expression of either wild-type or kinase-inactive Brk in Brk-null MDA-MB-435 cells activated ERK5 and conferred increased HGF-induced cell migration.

CONCLUSIONS

These results have identified Brk and ERK5 as important downstream effectors of Met signaling to cell migration. Targeting ERK5 kinase activity or inhibiting the formation of Brk/ERK5 complexes may provide an additional means of blocking cell migration associated with breast cancer progression to metastasis.

m-Calpain plays a critical role in cell migration enabling rear de-adhesion of adherent cells by cleaving structural components of the adhesion plaques. <em>Growth</em> <em>factors</em> and chemokines regulate <em>keratinocyte</em>, fibroblast, and endothelial cell migration by modulating m-calpain activity. <em>Growth</em> <em>factor</em> receptors activate m-calpain secondary to phosphorylation on serine 50 by ERK. Concurrently, activated m-calpain is localized to its inner membrane milieu by binding to phosphatidylinositol 4,5-bisphosphate (PIP(<em>2</em>)). Opposing this, CXCR3 ligands inhibit cell migration by blocking m-calpain activity secondary to a PKA-mediated phosphorylation in the C<em>2</em>-like domain. The failure of m-calpain activation in the absence of PIP(<em>2</em>) points to a key regulatory role, although whether this PIP(<em>2</em>)-mediated membrane localization is regulatory for m-calpain activity or merely serves as a docking site for ERK phosphorylation is uncertain. Herein, we report the effects of two CXCR3 ligands, CXCL11/IP-9/I-TAC and CXCL10/IP-10, on the EGF- and VEGF-induced redistribution of m-calpain in human fibroblasts and endothelial cells. The two chemokines block the tail retraction and, thus, the migration within minutes, preventing and reverting <em>growth</em> <em>factor</em>-induced relocalization of m-calpain to the plasma membrane of the cells. PKA phosphorylation of m-calpain blocks the binding of the protease to PIP(<em>2</em>). Unexpectedly, we found that this was due to membrane anchorage itself and not merely serine 50 phosphorylation, as the farnesylation-induced anchorage of m-calpain triggers a strong activation of this protease, leading notably to an increased cell death. Moreover, the ERK and PKA phosphorylations have no effect on this membrane-anchored m-calpain. However, the presence of PIP(<em>2</em>) is still required for the activation of the anchored m-calpain. In conclusion, we describe a novel mechanism of m-calpain activation by interaction with the plasma membrane and PIP(<em>2</em>) specifically, this phosphoinositide acting as a co<em>factor</em> for the enzyme. The phosphorylation of m-calpain by ERK and PKA by <em>growth</em> <em>factors</em> and chemokines, respectively, act in cells to regulate the enzyme only indirectly by controlling its redistribution.

OBJECTIVE

Type <em>2</em> diabetes and obesity are associated with increased risk of site-specific cancers. We have investigated whether metabolic alterations at the level of adipose-derived differentiating cells may affect specific phenotypes of breast cancer cells.

METHODS

Growth profiles of breast cancer cell lines were evaluated in co-cultures with differentiated adipocytes or their precursor cells and upon treatment with adipocyte conditioned media. Production and release of cytokines and growth factors were assessed by real-time RT-PCR and multiplex-based ELISA assays.

RESULTS

Co-cultures with either differentiated mouse 3T3-L1 or human mammary adipocytes increased viability of MCF-7 cells to a greater extent, when compared with their undifferentiated precursors. Adipocytes cultured in <em>2</em>5 mmol/l glucose were twofold more effective in promoting cell growth, compared with those grown in 5.5 mmol/l glucose, and activated mitogenic pathways in MCF-7 cells. Growth-promoting action was also enhanced when adipocytes were incubated in the presence of palmitate or oleate. Interestingly, 3T3-L1 and human adipocytes released higher amounts of keratinocyte-derived chemokine/IL-8, the protein 'regulated upon activation, normally T expressed, and secreted' (RANTES), and IGF-1, compared with their precursor cells. Their levels were reduced upon incubation with low glucose and enhanced by fatty acids. Moreover, both undifferentiated cells and differentiated adipocytes from obese individuals displayed about twofold higher IGF-1 release and MCF-7 cell growth induction than lean individuals. Finally, inhibition of the IGF-1 pathway almost completely prevented the growth-promoting effect of adipocytes on breast cancer cells.

CONCLUSIONS

IGF-1 release by adipocytes is regulated by glucose and fatty acids and may contribute to the control of cancer cell growth in obese individuals.

Hepatocyte <em>growth</em> <em>factor</em> (HGF) and <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) are among the most potent mitogens identified for alveolar type II epithelial cells and may have other important functions in repair of the alveolar epithelium in acute lung injury (ALI). However, neither <em>growth</em> <em>factor</em> has been identified in the distal air spaces or plasma of patients with ALI. The goals of this study were to determine: (1) whether HGF and KGF are present in pulmonary edema fluid from patients with ALI and control patients with hydrostatic pulmonary edema; (<em>2</em>) whether HGF and KGF are biologically active in pulmonary edema; and (3) whether HGF or KGF levels are associated with clinical outcome. Pulmonary edema and plasma samples were obtained within 48 h of onset of acute pulmonary edema requiring mechanical ventilation in <em>2</em>6 patients with ALI and 11 control patients with hydrostatic edema. HGF and KGF concentrations were measured with enzyme-linked immunosorbent assays (ELISAs). The median (<em>2</em>5th to 75th percentiles) concentration of HGF in pulmonary edema fluid was <em>2</em>1.4 (8.3 to 41.3) ng/ml in ALI and 6.6 (4.8 to 11.4) ng/ml in hydrostatic edema fluid (p < 0.01). The HGF concentration was 7-fold higher in the edema fluid than in the plasma of patients with ALI. In contrast, KGF was detected in low concentrations in edema fluid of patients with ALI and hydrostatic pulmonary edema; the concentration of KGF did not differ in ALI edema (0.6 [0.3 to <em>2</em>.1] ng/ml) and hydrostatic edema fluid (0.<em>2</em> [0.0 to <em>2</em>.6] ng/ml) (p = NS). HGF and KGF were partly purified from four edema-fluid samples by heparin-Sepharose chromatography. Partly purified edema fluids were potent stimuli of DNA synthesis in cultured rat type II alveolar cells; addition of neutralizing antibodies to HGF and KGF attenuated this increase in DNA synthesis by 66% and 53%, respectively. Interestingly, higher edema-fluid levels of HGF were associated with higher mortality in patients with ALI. These novel results show that HGF and KGF are active in the alveolar space early in ALI, probably mediating early events in lung repair, and that increased levels of HGF in edema fluid may have prognostic value early in ALI.

Alteration in the expression of <em>growth</em> <em>factors</em> is widely accepted as being one of several critical defects in the generation of the malignant cell. In the present study, 19 human metastatic melanoma cell lines were compared to 14 normal human foreskin melanocyte cell lines for the production of RNA transcripts specific for 11 different <em>growth</em> <em>factors</em>. Using the extremely sensitive technique of polymerase chain reaction to amplify <em>growth</em> <em>factor</em>-specific complementary DNAs, we analyzed the following: transforming <em>growth</em> <em>factor</em> (TGF) types alpha, beta 1, beta <em>2</em>, and beta 3, acidic (a) fibroblast <em>growth</em> <em>factor</em> (FGF), basic (b) FGF, FGF-5, <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), HST, and platelet-derived <em>growth</em> <em>factor</em> (PDGF) types A and B. There were clear distinctions among the patterns of <em>growth</em> <em>factor</em> RNA expression by normal melanocytes and malignant melanoma cells. The prototypic melanocyte pattern of expression included TGF beta 1, TGF beta 3, and KGF. A subset of melanocyte cell lines also expressed PDGFA transcripts. In contrast, melanoma cells characteristically expressed RNA transcripts of TGF beta 1, TGF beta <em>2</em>, TGF beta 3, TGF alpha, bFGF, KGF, and PDGFA. Subsets of melanoma cell lines also expressed aFGF, FGF-5, and PDGFB. The results presented indicated that TGF beta <em>2</em>, TGF alpha, and bFGF may be particularly important in melanomagenesis and that these, as well as FGF-5, aFGF, and PDGFB, can be used as markers of transformation in this tumor type.

The bek gene encodes a member of the high-affinity fibroblast <em>growth</em> <em>factor</em> receptor family. The BEK/FGFR-<em>2</em> receptor is a membrane-spanning tyrosine kinase with the typical features of FGF receptors. We have cloned a murine bek cDNA and expressed it in receptor-negative Chinese hamster ovary cells and in 3<em>2</em>D myeloid cells. The BEK receptor expressed in Chinese hamster ovary cells binds acidic FGF, basic FGF, and Kaposi FGF equally well but does not bind <em>keratinocyte</em> <em>growth</em> <em>factor</em> or FGF-5 appreciably. Upon treatment with basic FGF or Kaposi FGF, the BEK receptor is phosphorylated and a mitogenic response is achieved. Heparan sulfate proteoglycans have been shown to play an obligate role in basic FGF binding to the high-affinity FLG receptor. Unlike the BEK-expressing Chinese hamster ovary cells, 3<em>2</em>D cells expressing the BEK receptor require the addition of exogenous heparin in order to grow in the presence of basic FGF or Kaposi FGF. We show that the addition of heparin greatly enhances the binding of radio-labeled basic FGF to the receptor. Thus the BEK receptor, like FLG, also requires an interaction with heparan sulfate proteoglycans to facilitate binding to its ligands.

The differentiation of isolated human cytotrophoblast cells has been studied by staining cells with anti-desmoplakin antibodies, to reveal cell boundaries, and with anti-nuclear antibodies, to reveal nuclei. During the first <em>2</em>4 h after plating in Ham's/Waymouth medium, mononucleated cytotrophoblast cells began to spread and aggregate, forming colonies. This was accompanied by progressive changes in the pattern of desmoplakin staining. In single cells, desmoplakin was dispersed throughout the cytoplasm. As cells aggregated, desmoplakin was redistributed and formed linear, punctate arrays at regions of cell-cell contact, consistent with desmosome formation. A pavement-like staining pattern was maintained even at 5 days. Double staining for desmoplakin and nuclei revealed that most cells within colonies were mononucleated. When plated in a <em>growth</em> medium originally formulated for <em>keratinocytes</em>, cytotrophoblast cells aggregated and formed desmosomes normally. However, after 48 h, cell diameters were increased and nuclei changed from being evenly distributed to forming clusters within large cells, consistent with syncytiotrophoblast formation. While cells grown in Ham's/Waymouth medium for <em>2</em> days could be induced to differentiate by switching to <em>keratinocyte</em> <em>growth</em> medium, cells cultured for 5 days before switching were resistant to the differentiation-inducing effects of the <em>keratinocyte</em> medium. Desmosome-type junctions within colonies of trophoblast cells were unstable and, even after 5 days in culture, could be disrupted by lowering the extracellular Ca<em>2</em>+ concentration. While syncytiotrophoblast formation in <em>keratinocyte</em> <em>growth</em> medium (which contains epidermal <em>growth</em> <em>factor</em>, insulin and hydrocortisone) was accompanied by a 15- to <em>2</em>0-fold increase in chorionic gonadotropin secretion, syncytiotrophoblast formation occurred to a similar extent in <em>keratinocyte</em> basal medium (which does not contain these <em>factors</em>) but with only a twofold increase in chorionic gonadotropin release. These results support the notion that biochemical and morphological differentiation of trophoblast are independent events.

Chronic inflammation is an important underlying condition for ovarian tumor development, <em>growth</em> and progression. Since chemokine networks are activated by inflammation, patterns of chemokine gene expression were investigated in ovarian cancer cells. Chemokine specific microarrays were performed after mouse (ID8) and human (SKOV-3) ovarian surface epithelial cancer cells were exposed to the inflammatory agent bacterial endotoxin lipopolysaccharide (LPS, 10 microg/ml) and pro-inflammatory cytokines interleukin-1beta (IL-1, 10 ng/ml) and tumor necrosis <em>factor</em>-alpha (TNF, 10 ng/ml). In the mouse ID8 cells, LPS, IL-1 and TNF led to robust upregulation of <em>keratinocyte</em> chemoattractant (KC) chemokines CXCL1/<em>2</em>, mouse homologues of human <em>growth</em>-regulated oncogenes (GRO). Other chemokines, interferong inducible protein (IP)-10 (CXCL10), CCL7 and CCL<em>2</em>0 were moderately upregulated. ID8 cells constitutively expressed CXCL16 and CCL<em>2</em>, but only CCL<em>2</em> expression was enhanced by LPS, IL-1 and TNF. In the human SKOV-3 cells, LPS had no effect on chemokines expression due to the absence of the LPS receptor, toll-like receptor 4 (TLR4). However, IL-1 and TNF induced GROalpha/beta (CXCL1/<em>2</em>) in human SKOV-3 cells in a similar manner as observed with mouse ID8 cells. In SKOV-3 cells, IL-8 (CXCL8) was highly expressed and other chemokines GROgamma (CXCL3) and CCL<em>2</em>0 were moderately expressed in response to IL-1 and TNF. The nuclear <em>factor</em>-kappaB (NF-kappaB) is a known mediator of cytokine and chemokines signaling. The NFkappaB inhibitor BAY 11-708<em>2</em> attenuated expression of inflammatory-induced chemokines in the mouse and human ovarian cancer cells. Taken together, the results indicate that KC/GRO chemokines are the principal chemokines induced by LPS and pro-inflammatory cytokines IL-1 and TNF via NFkappaB signaling in ovarian surface epithelial cancer cells.