Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis

Background Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. Methods The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. Results We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury. Conclusion In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.

Effectiveness of a 308-nm excimer laser in treatment of vitiligo: a review

Vitiligo is a relatively common acquired disorder, characterized by progressive loss of melanocytes from the epidermis and the epidermal appendages. The disease is associated with considerable morbidity because of a major impact on the quality of life. The treatment for vitiligo is generally unsatisfactory and challenging. There are a variety of therapeutic possibilities including topical corticosteroids, topical calcineurin inhibitors, as well as phototherapy with Psoralen plus UVA (PUVA), narrow-band UVB, and a 308-nm excimer laser and/or lamps. Furthermore, surgical methods encompass grafting and transplantation while depigmentation treatments and psychological support may also be considered. The objective is to assess the effect of the 380-nm excimer laser in the treatment of vitiligo based on the available studies and case series. We searched the relevant literature about vitiligo and excimer laser published between 1990 and 2012 using the MEDLINE database. We reviewed all relevant articles about 308-nm excimer laser and light sources assessing their efficacy in the management of vitiligo as well as their side effects. The value of combination treatment methods was also analyzed. The available studies provide strong evidence that the excimer laser represents the most effective approach to treat vitiligo compared to ordinary phototherapy. Excimer laser is relatively safe and effective for localized disease. UV-sensitive areas respond best as well as a short duration of the disease. More frequent treatments achieve better results. Compared to other treatment modalities, the excimer laser most likely constitutes the treatment of choice for localized vitiligo. Its efficacy can be further improved in combination with other therapies such as corticosteroids, pimecrolimus, or tacrolimus.

Phosphatase and tensin homolog regulates stability and activity of EphB1 receptor

Deregulation of receptor tyrosine kinases (RTKs) is linked to a broad range of cancers, stressing the necessity of studying their regulatory pathways. We and others demonstrated previously that c-Cbl is necessary for the lysosomal degradation of erythropoietin-producing hepatocellular B1 (EphB1) carcinoma and epidermal growth factor receptor (EGFR) RTKs. Moreover, the tumor suppressor phosphatase and tensin homolog (PTEN) was shown to modulate c-Cbl-dependent EGFR degradation. We therefore investigated the involvement of PTEN in EphB1 signaling and degradation. We used PTEN mutants, PTEN, and NHERF1 small interfering RNA in CHO-EphB1 and SW480 cells endogenously expressing EphB1 to delineate EphB1-PTEN interactions. PTEN was constitutively associated with c-Cbl, protecting it from degradation. EphB1 stimulation triggered ∼50% serine-threonine PTEN dephosphorylation and PTEN-Cbl complex disruption, a process requiring PTEN protein phosphatase activity. Both proteins independently translocated to EphB1, with PTEN in association with the scaffold protein NHERF1. Biologically, PTEN lipid phosphatase activity impairs EphB1-dependent cell adhesion and chemotaxis. This study demonstrates for the first time in mammalian cells that the Eph receptor and PTEN associate and influence their signaling. Moreover, it contributes to the emerging concept that PTEN regulates expression of RTKs through modulation of their degradation. Finally, it reveals a new role for PTEN protein phosphatase activity involved in this process.

Phosphoinositide 3-kinase C2β regulates RhoA and the actin cytoskeleton through an interaction with Dbl

The regulation of cell morphology is a dynamic process under the control of multiple protein complexes acting in a coordinated manner. Phosphoinositide 3-kinases (PI3K) and their lipid products are widely involved in cytoskeletal regulation by interacting with proteins regulating RhoGTPases. Class II PI3K isoforms have been implicated in the regulation of the actin cytoskeleton, although their exact role and mechanism of action remain to be established. In this report, we have identified Dbl, a Rho family guanine nucleotide exchange factor (RhoGEF) as an interaction partner of PI3KC2β. Dbl was co-immunoprecipitated with PI3KC2β in NIH3T3 cells and cancer cell lines. Over-expression of Class II phosphoinositide 3-kinase PI3KC2β in NIH3T3 fibroblasts led to increased stress fibres formation and cell spreading. Accordingly, we found high basal RhoA activity and increased serum response factor (SRF) activation downstream of RhoA upon serum stimulation. In contrast, the dominant-negative form of PI3KC2β strongly reduced cell spreading and stress fibres formation, as well as SRF response. Platelet-derived growth factor (PDGF) stimulation of wild-type PI3KC2β over-expressing NIH3T3 cells strongly increased Rac and c-Jun N-terminal kinase (JNK) activation, but failed to show similar effect in the cells with the dominant-negative enzyme. Interestingly, epidermal growth factor (EGF) and PDGF stimulation led to increased extracellular signal-regulated kinase (Erk) and Akt pathway activation in cells with elevated wild-type PI3KC2β expression. Furthermore, increased expression of PI3KC2β protected NIH3T3 from detachment-dependent death (anoikis) in a RhoA-dependent manner. Taken together, these findings suggest that PI3KC2β modulates the cell morphology and survival through a specific interaction with Dbl and the activation of RhoA.

Reduced nuclear translocation of nuclear factor (NF)-kappaB p65 in the footpad epidermis of dogs infected with distemper virus

Infection of canine footpads with the canine distemper virus (CDV) can cause massive epidermal thickening (hard pad disease), as a consequence of increased proliferation of keratinocytes and hyperkeratosis. Keratinocytes of canine footpad epidermis containing detectable CDV nucleoprotein antigen and CDV mRNA were shown previously to have increased proliferation indices. Because various proteins that play a role in the proliferation of epidermal cells are viral targets, the potential participation of such proteins in CDV-associated keratinocyte proliferation was investigated. Transforming growth factor-alpha (TGF-alpha), cell cycle regulatory proteins p21, p27 and p53, and nuclear factor (NF)-kappaB transcription factor components p50 and p65 were studied in the footpad epidermis from the following groups of dogs inoculated with CDV: group 1, consisting of seven dogs with clinical distemper and CDV in the footpad epidermis; group 2, consisting of four dogs with clinical distemper but no CDV in the footpad epidermis; group 3, consisting of eight dogs with neither clinical distemper nor CDV in the footpad epithelium. Group 4 consisted of two uninoculated control dogs. The expression of TGF-alpha, p21, p27 and p53, and p50 in the basal layer, lower and upper spinous layers, and in the granular layer did not differ statistically between CDV-positive (group 1) and CDV-negative (groups 2-4) footpad epidermis. However, there were differences in the levels of nuclear and cytoplasmic p65 expression between group 1 dogs and the other three groups. Thus, footpads from group 1 dogs had more keratinocytes containing p65 in the cytoplasm and, conversely, fewer nuclei that were positive for p65. These findings indicate that p65 translocation into the nucleus is reduced in CDV-infected footpad epidermis. Such decreased translocation of p65 may help to explain increased keratinocyte proliferation in hard pad disease and suggests interference of CDV with the NF-kappaB pathway.

Black hair follicular dysplasia in Large Münsterländer dogs: clinical, histological and ultrastructural features

Four Large Münsterländer cross-bred dogs affected with black hair follicular dysplasia (BHFD) and one unaffected control littermate were observed, and skin was sampled weekly over the first 19 weeks of life. Affected dogs were born with silvery grey hair, a consequence of melanin clumping in the hair shafts. Hair bulb melanocytes were densely pigmented, and contained abundant stage IV melanosomes but adjacent matrix keratinocytes lacked melanosomes. Melanin clumping was not prominent in epidermal melanocytes in the haired skin but occurred in the foot pads. Follicular changes progressed from bulbar clumping, clumping in the isthmus/infundibulum and finally to dysplastic hair shafts. Alopecia developed progressively in pigmented areas. Silver-grey hair, melanin clumping, accumulation of stage IV melanosomes within melanocytes and insufficient melanin transfer to adjacent keratinocytes are also classic features of human Griscelli syndrome. The underlying cause in Griscelli syndrome is a defect of melanocytic intracellular transport proteins leading to inadequate and disorganized melanosome transfer to keratinocytes with resultant melanin clumping. In view of the correlation in the phenotype, histology and ultrastructure between both disorders, a defect in intracellular melanosome transport is postulated as the pathogenic mechanism in BHFD.

Keratin variants associate with progression of fibrosis during chronic hepatitis C infection

Keratins 8 and 18 (K8/K18) protect the liver from various forms of injury. Studies of liver explants from a large cohort of U.S. patients showed that K8/K18 mutations confer a risk to developing end-stage liver diseases, though which diseases are preferentially involved is unknown. We tested the hypothesis that K8/K18 variants are associated with chronic hepatitis C (CHC) and that their presence correlates with progression of fibrosis. Genomic DNA was isolated from peripheral blood of a well-characterized German cohort of 329 patients with CHC infection. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Our findings showed: (1) amino acid altering keratin heterozygous variants in 24 of 329 CHC patients (7.3%) and non-coding heterozygous variants in 26 patients (7.8%), and (2) 3 new exonic K8 variants (T26R/G55A/A359T); 6 novel non-coding variants and one K18 coding variant (K18 S230T; 2 patients). The most common variants were K8 R341H (10 patients), K8 G62C (6 patients) and K8 I63V (4 patients). A novel and exclusive association of an intronic KRT8 IVS7+10delC deletion in all 10 patients with K8 R341H was observed. Notably, there was a significant association of exonic, but not of intronic K8 variants with increased fibrosis. In conclusion, previously described and novel K8 variants are present in a German population and collectively associate with progression of fibrosis in CHC infection. The unique 100% segregation of the most common K8 variant, R341H, with an intronic deletion suggests that one of these two genetic changes might lead to the other.

Consequences of depleted SERCA2-gated calcium stores in the skin

Sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) pumps belong to the family of Ca2+-ATPases responsible for the maintenance of calcium in the endoplasmic reticulum. In epidermal keratinocytes, SERCA2-controlled calcium stores are involved in cell cycle exit and onset of terminal differentiation. Hence, their dysfunction was thought to provoke impaired keratinocyte cohesion and hampered terminal differentiation. Here, we assessed cultured keratinocytes and skin biopsies from a canine family with an inherited skin blistering disorder. Cells from lesional and phenotypically normal areas of one of these dogs revealed affected calcium homeostasis due to depleted SERCA2-gated stores. In phenotypically normal patient cells, this defect compromised upregulation of p21(WAF1) and delayed the exit from the cell cycle. Despite this abnormality it failed to impede the terminal differentiation process in the long term but instead coincided with enhanced apoptosis and appearance of chronic wounds, suggestive of secondary mutations. Collectively, these findings provide the first survey on phenotypic consequences of depleted SERCA-gated stores for epidermal homeostasis that explain how depleted SERCA2 calcium stores provoke focal lesions rather than generalized dermatoses, a phenotype highly reminiscent of the human genodermatosis Darier disease.

Pemphigus vulgaris identifies plakoglobin as key suppressor of c-Myc in the skin

The autoimmune disease pemphigus vulgaris (PV) manifests as loss of keratinocyte cohesion triggered by autoantibody binding to desmoglein (Dsg)3, an intercellular adhesion molecule of mucous membranes, epidermis, and epidermal stem cells. Here we describe a so far unknown signaling cascade activated by PV antibodies. It extends from a transient enhanced turn over of cell surface-exposed, nonkeratin-anchored Dsg3 and associated plakoglobin (PG), through to depletion of nuclear PG, and as one of the consequences, abrogation of PG-mediated c-Myc suppression. In PV patients (6/6), this results in pathogenic c-Myc overexpression in all targeted tissues, including the stem cell compartments. In summary, these results show that PV antibodies act via PG to abolish the c-Myc suppression required for both maintenance of epidermal stem cells in their niche and controlled differentiation along the epidermal lineage. Besides a completely novel insight into PV pathogenesis, these data identify PG as a potent modulator of epithelial homeostasis via its role as a key suppressor of c-Myc.

Grover's disease induced by cetuximab

A 71-year-old man exhibited an acute acneiform rash affecting the face and the upper trunk about 2 weeks after starting cetuximab, an epidermal growth factor (EGF) receptor antagonist treatment for metastatic colon cancer. The skin eruption faded after stopping cetuximab and applying topical corticosteroids. The reexposure to cetuximab 3 weeks later provoked a more extended relapse of the skin rash, which then clinically and histologically corresponded to transient acantholytic dermatosis . While the acneiform cutaneous side effects of the EGF receptor antagonists are interpreted as a result of the direct interference with pilosebaceous follicle homeostasis, in this case an acrosyringium-related pathogenesis might be postulated. Applying topical corticosteroids and emollients, the cetuximab therapy could be pursued.

[Molecular targets in colon cancer]

Colorectal cancer is the second leading cause of cancer death in Switzerland. The nihilism that dominated the treatment of these patients for decades has been replaced by a measure of enthusiasm, given recent therapeutic advances. New anticancer drugs such as irinotecan and oxaliplatin have changed the standard chemotherapy treatment of metastatic colorectal cancer. However, the real hype has come from molecular targeted therapy. Identification of cellular processes characteristic of colon cancer has permitted therapeutic targeting with favorable therapeutic index. Inhibition of the epidermal growth factor receptor in the clinic has provided proof of principle that interruption of signal transduction cascades in patients has therapeutic potential. Angiogenesis, especially the vascular endothelial growth factor pathway, has been proven to be another highly successful molecular target. In this article, we will review molecular targets, which are under active clinical investigation in colon cancer.

Expression of HB-EGF by retinal pigment epithelial cells in vitreoretinal proliferative disease

The heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated in wound-healing processes of various tissues. However, it is not known whether HB-EGF may represent a factor implicated in overstimulated wound-healing processes of the retina during proliferative retinopathies. Therefore, we investigated whether human retinal pigment epithelial (RPE) cells, which are crucially involved in proliferative retinopathies, express and respond to HB-EGF. RPE cells express mRNAs for various members of the EGF-related growth factor family, among them for HB-EGF, as well as for the EGF receptors ErbB1, -2, -3, and -4. The gene expression of HB-EGF is stimulated in the presence of transforming and basic fibroblast growth factors and by oxidative stress and is suppressed during chemical hypoxia. Exogenous HB-EGF stimulates proliferation and migration of RPE cells and the gene and protein expression of the vascular endothelial growth factor (VEGF). HB-EGF activates at least three signal transduction pathways in RPE cells including the extracellular signal-regulated kinases (involved in the proliferation-stimulating action of HB-EGF), p38 (mediates the effects on chemotaxis and secretion of VEGF), and the phosphatidylinositol-3 kinase (necessary for the stimulation of chemotaxis). In epiretinal membranes of patients with proliferative retinopathies, HB-EGF immunoreactivity was partially colocalized with the RPE cell marker, cytokeratins; this observation suggests that RPE cell-derived HB-EGF may represent one factor that drives the uncontrolled wound-healing process of the retina. The stimulating effect on the secretion of VEGF may suggest that HB-EGF is also implicated in the pathological angiogenesis of the retina.

Tie2 receptor expression and phosphorylation in cultured cells and mouse tissues

Accumulating experimental evidence indicates that endothelial cell growth and blood vessel morphogenesis are processes that are governed by the activity of specifically expressed receptor tyrosine kinases (RTKs). We have used two new rat monoclonal antibodies (mAbs) to study the expression and phosphorylation of one such receptor, mouse Tie2 (tyrosine kinase that contains immunoglobulin-like loops and epidermal-growth-factor-similar domains 2]), in transfected cells, endothelioma cell lines and mouse tissues. The Tie2 receptor was found to be constitutively autophosphorylated when over-expressed in COS7 cells. In contrast, the endogenous Tie2 protein was not phosphorylated in endothelioma cell lines. However, in these cell lines, Tie2 could be induced to become tyrosine phosphorylated, and this activation was found to be independent of Tie1. Studying Tie2 receptor activity during angiogenesis in mouse development, the receptor was only weakly phosphorylated in the early postnatal mouse brain whereas a stronger activation could be detected in mouse embryos at day 10.5 post coitum.

Treatment of premenopausal women with early breast cancer: old challenges and new opportunities

Breast cancer occurring in women before the age of menopause continues to be a major medical and psychological challenge. Endocrine therapy has emerged as the mainstay of adjuvant treatment for women with estrogen receptor-positive tumours. Although the suppression of ovarian function (by oophorectomy, irradiation of the ovaries or gonadotropin releasing factor analogues) is effective as adjuvant therapy if used alone, its value has not been proven after chemotherapy. This is presumably because of the frequent occurrence of chemotherapy-induced amenorrhoea. Tamoxifen reduces the risk of recurrence by approximately 40%, irrespective of age and the ovarian production of estrogens. The worth of ovarian function suppression in combination with tamoxifen is unproven and is being investigated in an intergroup randomised clinical trial (SOFT [Suppression of Ovarian Function Trial]). Aromatase inhibitors are more effective than tamoxifen in postmenopausal women but are only being investigated in younger patients. The use of chemotherapies is identical in younger and older patients; however, at present the efficacy of chemotherapy in addition to ovarian function suppression plus tamoxifen is unknown in premenopausal patients with endocrine responsive disease. 'Targeted' therapies such as monoclonal antibodies to human epidermal growth factor receptor (HER)-2, HER1 and vascular endothelial growth factor, 'small molecule' inhibitors of tyrosine kinases and breast cancer vaccines are rapidly emerging. Their use depends on the function of the targeted pathways and is presently limited to clinical trials. Premenopausal patients are best treated in the framework of a clinical trial.

An open-label, noncomparative phase II trial to evaluate the efficacy and safety of docetaxel in combination with gefitinib in patients with hormone-refractory metastatic prostate cancer

BACKGROUND: Prostate cancer is the most common type of cancer in men, however, therapeutic options are limited. 50-90% of hormone-refractory prostate cancer cells show an overexpression of epidermal growth factor receptor (EGFR), which may contribute to uncontrolled proliferation and resistance to chemotherapy. In vitro, gefitinib, an orally administered tyrosine kinase inhibitor, has shown a significant increase in antitumor activity when combined with chemotherapy. PATIENTS AND METHODS: In this phase II study, the safety and efficacy of gefitinib in combination with docetaxel, a chemotherapeutic agent commonly used for prostate cancer, was investigated in patients with hormone-refractory prostate cancer (HRPC). 37 patients with HRPC were treated continuously with gefitinib 250 mg once daily and docetaxel 35 mg/m2 i.v. for up to 6 cycles. PSA response, defined as a =50% decrease in serum PSA compared with trial entry, was the primary efficacy parameter. PSA levels were measured at prescribed intervals. RESULTS: The response rate and duration of response were consistent with those seen with docetaxel monotherapy. The combination of docetaxel and gefitinib was reasonably well tolerated in this study. CONCLUSION: Future studies should investigate whether patients with specific tumor characteristics, e.g. EGFR protein overexpression, respond better to gefitinib than patients without, leading to a more customized therapy option.