Heparanase is preferentially expressed in human psoriatic lesions and induces development of psoriasiform skin inflammation in mice

Heparanase is the sole mammalian endoglycosidase that selectively degrades heparan sulfate, the key polysaccharide associated with the cell surface and extracellular matrix of a wide range of tissues. Extensively studied for its capacity to promote cancer progression, heparanase enzyme was recently implicated as an important determinant in several inflammatory disorders as well. Applying immunohistochemical staining, we detected preferential expression of heparanase by epidermal keratinocytes in human psoriatic lesions. To investigate the role of the enzyme in the pathogenesis of psoriasis, we utilized heparanase transgenic mice in a model of 12-O-tetradecanoyl phorbol 12-myristate 13-acetate-induced cutaneous inflammation. We report that over-expression of the enzyme promotes development of mouse skin lesions that strongly recapitulate the human disease in terms of histomorphological appearance and molecular/cellular characteristics. Importantly, heparanase of epidermal origin appears to facilitate abnormal activation of skin-infiltrating macrophages, thus generating psoriasis-like inflammation conditions, characterized by induction of STAT3, enhanced NF-κB signaling, elevated expression of TNF-α and increased vascularization. Taken together, our results reveal, for the first time, involvement of heparanase in the pathogenesis of psoriasis and highlight a role for the enzyme in facilitating abnormal interactions between immune and epithelial cell subsets of the affected skin. Heparanase inhibitors (currently under clinical testing in malignant diseases) could hence turn highly beneficial in psoriatic patients as well.     

Role of G3BP1 in glucocorticoid receptor-mediated microRNA-15b and microRNA-23a biogenesis in endothelial cells

MicroRNAs (miRNAs) are a family of non-coding RNAs that play crucial roles in regulating various normal cellular responses. Recent studies revealed that the canonical miRNA biogenesis pathway is subject to sophisticated regulation. Hormonal control of miRNA biogenesis by androgen and estrogen has been demonstrated, but the direct effects of the glucocorticoid receptor (GR) on miRNA biogenesis are unknown. This study revealed the role of GR in miRNA maturation. We showed that two GR agonists, dexamethasone and ginsenoside-Rg1 rapidly suppressed the expression of mature miR-15b, miR-23a, and miR-214 in human endothelial cells. RNA pulldown coupled with proteomic analysis identified GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) as one of the RNA-binding proteins mediating GR-regulated miRNA maturation. Activated GR induced phosphorylation of v-AKT Murine Thymoma Viral Oncogene Homologue (AKT) kinase, which in turn phosphorylated and promoted nuclear translocation of G3BP1. The nuclear G3BP1 bound to the G3BP1 consensus sequence located on primary miR-15b~16-2 and miR-23a~27a~24-2 to inhibit their maturation. The findings from this study have advanced our understanding of the non-genomic effects of GR in the vascular system.     

Cellular and functional evidence for a protective action of neurosteroids against vincristine chemotherapy-induced painful neuropathy

Painful neuropathy is a major side-effect limiting cancer chemotherapy. Therefore, novel strategies are required to suppress the neuropathic effects of anticancer drugs without altering their chemotherapeutic effectiveness. By combining biochemical, neuroanatomical/neurochemical, electrophysiological and behavioral methods, we demonstrated that progesterone-derived neurosteroids including 5α-dihydroprogesterone and 3α,5α-tetrahydroprogesterone suppressed neuropathic symptoms evoked in naive rats by vincristine. Neurosteroids counteracted vincristine-induced alterations in peripheral nerves including 2′,3′-cyclic nucleotide 3′-phosphodiesterase, neurofilament-200 kDa and intraepidermal nerve fiber repression, nerve conduction velocity, and pain transmission abnormalities (allodynia/hyperalgesia). In skin-tumor rats generated with carcinosarcoma-cells, vincristine, which suppressed the skin tumor and restored normal blood concentration of vascular endothelial growth factor (VEGF), reproduced neuropathic side-effects. Administered alone, neurosteroids did not affect the tumor and VEGF level. Combined with vincristine, neurosteroids preserved vincristine anti-tumor action but counteracted vincristine-induced neural side-effects. Together, these results provide valuable insight into the cellular and functional mechanisms underlying anticancer drug-induced neuropathy and suggest a neurosteroid-based strategy to eradicate painful neuropathy.     

Primary culture of dispersed skin epidermal cells of rainbow trout Oncorhynchus mykiss Walbaum

This is the first report on a primary culture of dispersed skin epidermal cells of rainbow trout Oncorhynchus mykiss Walbaum. These primary cells revealed a low seeding efficiency after 3 days (11.6 ± 4.6%), whereas subcultured cells had a higher seeding efficiency at the same time point (75.5 ± 34.0%) and increased in cell number (150 – 200% of seeded cells after 20 to 30 days). The cells were characterized applying histological, immunocytochemical and ultrastructural methods. The culture consisted of undifferentiated keratinocytes. Mucous cells as well as differentiated epithelial cells were absent. To date the cells were cultured for maximally 9 passages and 402 days and therefore provide the possibility for long-term studies. Received 31 March 1998; received after revision 14 July 1998; accepted 14 July 1998     

Number of glucocorticoid receptors in lymphocytes and their sensitivity to hormone action

The study demonstrated a decreased level of glucocorticoid receptors (GR) in peripheral blood lymphocytes from hypercholesterolemic subjects, and an elevated level in patients with acute myocardial infarction. In the lymphocytes with a high GR number, dexamethasone inhibited [3H]-thymidine and [3H]-acetate incorporation into DNA and cholesterol, respectively, in the same manner as in the control cells. On the other hand, a decreased GR number resulted in a less efficient dexamethasone inhibition of the incorporation of labeled compounds. These data showed that the sensitivity of lymphocytes to glucocorticoids changed only with a decrease of GR level.     

Dissection of the molecular mechanisms that control the nuclear accumulation of transport factors importin-α and CAS in stressed cells

The physiological state of eukaryotic cells controls nuclear trafficking of numerous cargos. For example, stress results in the inhibition of classical protein import, which is characterized by the redistribution of several transport factors. As such, importin-alpha and cellular apoptosis susceptibility protein (CAS) accumulate in nuclei of heat-shocked cells; however, the mechanisms underlying this relocation are not fully understood. We now show that heat upregulates the initial docking of importin-alpha at the nuclear envelope and stimulates the translocation of CAS into the nuclear interior. Moreover, heat exposure compromises the exit of importin-alpha from nuclei and drastically increases its retention in the nucleoplasm, whereas CAS nuclear exit and retention are less affected. Taken together, our results support the idea that heat shock regulates importin-alpha and CAS nuclear accumulation at several levels. The combination of different stress-induced changes leads to the nuclear concentration of both transport factors in heat-stressed cells.     

Macrophages derived from bone marrow modulate differentiation of myeloid dendritic cells

Dendritic cells (DC) are specialized antigen-presenting cells. Bone marrow monocytes have been widely used to generate murine myeloid DC. We found that mouse macrophages derived from bone marrow CD11b⁺ monocytes influenced the differentiation of these precursors into DC. Modulation of differentiation was demonstrated by the down-regulation of CD11c, CD40, and CD86 expression and by IL-12 production. DC differentiated in the presence of conditioned medium from bone marrow-derived macrophage culture (MCM) had impaired ability to stimulate proliferation of, and IFN- γ production by, allogeneic CD4⁺ T cells. This inhibition of DC differentiation was mainly mediated by secretory products from macrophages but not by cell-cell contact. MCM contained higher concentrations of macrophage-colony-stimulating factor (M-CSF), IL-10, and TGF- β1, whereas IL-6 remained unchanged compared with conditioned medium from fresh monocytes. M-CSF may be the major mediator in MCM inhibiting DC differentiation. This study demonstrates an important influence of bone marrow-derived macrophages on DC precursors during DC differentiation. Received 12 September 2006; received after revision 20 October 2006; accepted 13 November 2006     

Heat evolution of cultured human keratinocytes

The heat production of normal and transformed human epidermal keratinocytes precultured in Petriperm tissue culture dishes was measured calorimetrically. For this purpose, the membrane at the bottom of the culture dish was cut out aseptically and put into a microcalorimeter vessel with the cell layer inwards. A continuous heat output of (83 +/- 12) pW/cell was measured for normal keratinocytes from a confluent primary culture. A value of (134 +/- 35) pW/cell was obtained when the transformed keratinocyte line SV-K14 was used. The method described in this paper is simple, leads to reproducible results, and can be easily adapted to the calorimetric study of other mammalian cells in vitro.     

Physiological roles of glycerol-transporting aquaporins: the aquaglyceroporins

A subclass of aquaporin (AQP) water channels, termed aquaglyceroporins, are also able to transport glycerol and perhaps urea and other small solutes. Although extensive data exist on the physiological roles of aquaporin-facilitated water transport, until recently the biological significance of glycerol transport by the mammalian aquaglyceroporins has been unknown. There is now compelling evidence for involvement of aquaglyceroporin- facilitated glycerol transport in skin hydration and fat cell metabolism. Mice deficient in AQP3 have dry skin, reduced skin elasticity and impaired epidermal biosynthesis. Mice lacking AQP7 manifest progressive adipocyte fat accumulation and hypertrophy. These skin and fat phenotypes are attributable to impaired glycerol transport. A potential implication of these findings is the possibility of modulation of aquaglyceroporin expression or function in the therapy of skin diseases and obesity. Received 20 January 2006; received after revision 21 February 2006; accepted 20 March 2006     

Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents

The isolation of human epidermal stem cells is critical for their clinical applications. In the present study, we isolated three populations of epidermal keratinocytes according to their ability to adhere to collagen type IV: i.e., rapidly adhering (RA), slowly adhering (SA), and non-adhering (NA) cells. The aim of this study was to characterize RA cells and to investigate the possibility of using these cells for epidermis reconstruction. To identify RA cells, flow cytometric analysis was performed using anti-₆ integrin and anti-CD71 antibodies. RA cells express high levels of ₆ integrin and low levels of CD71, which are considered as markers of an epidermal stem cell nature. Furthermore, electron microscopy showed that RA cells are small and have a high nuclear to cytoplasmic ratio, whereas SA and NA cells have well-developed cellular organelles and abundant tonofilaments. Western blot analysis showed that RA cells are slow cycling and express p63, a putative epidermal stem cell marker, whereas SA and NA cells express c-Myc, which is known to regulate stem cell fate. To compare epidermal regenerative abilities, skin equivalents (SEs) were made using RA, SA, and NA cells. The epidermis constructed from RA cells was well formed compared to those formed from SA or NA cells. In addition, only SEs with RA cells expressed ₆ integrin and ₁ integrin at the basal layer. These results indicate that RA cells represent epidermal stem cells and are predominately comprised of stem cells. Therefore, the isolation of RA cells using a simple technique offers a potential route to their clinical application, because they are easily isolated and provide a high yield of epidermal stem cells.Received 2 July 2004; received after revision 20 August 2004; accepted 10 September 2004     

Functions of skin-resident γδ T cells

The murine epidermis contains resident T cells that express a canonical γδ TCR and arise from fetal thymic precursors. These cells are termed dendritic epidermal T cells (DETC) and use a TCR that is restricted to the skin in adult animals. DETC produce low levels of cytokines and growth factors that contribute to epidermal homeostasis. Upon activation, DETC can secrete large amounts of inflammatory molecules which participate in the communication between DETC, neighboring keratinocytes and langerhans cells. Chemokines produced by DETC may recruit inflammatory cells to the epidermis. In addition, cell–cell mediated immune responses also appear important for epidermal–T cell communication. Information is provided which supports a crucial role for DETC in inflammation, wound healing, and tumor surveillance.     

Heat evolution of cultured human keratinocytes

Summary The heat production of normal and transformed human epidermal keratinocytes precultured in Petriperm ^tm tissue culture dishes was measured calorimetrically. For this purpose, the membrane at the bottom of the culture dish was cut out aseptically and put into a microcalorimeter vessel with the cell layer inwards. A continuous heat output of (83±12) pW/cell was measured for normal keratinocytes from a confluent primary culture. A value of (134±35) pW/cell was obtained when the transformed kerationocyte line SV-K14 was used. The method described in this paper is simple, leads to reproducible results, and can be easily adapted to the calorimetric study of other mammalian cells in vitro.     

Heavy incorporation of3H-prostaglandin F2α in the neoplastic cells as revealed by autoradiographic studies

Summary A marked uptake (9-fold) of the³H-PGF₂ was found specifically over heterochromatin in the nuclei of neoplastic cells. Lower but significant uptakes of³H-PGF₂ were also found in the nuclei of control epidermal cells, which indicate the presence of nuclear receptors in the epidermal neoplastic cells.     

Absence of a robust innate immune response in rat neurons facilitates persistent infection of Borna disease virus in neuronal tissue

Borna disease virus (BDV) persistently infects neurons of the central nervous system of various hosts, including rats. Since type I IFN-mediated antiviral response efficiently blocks BDV replication in primary rat embryo fibroblasts, it has been speculated that BDV is not effectively sensed by the host innate immune system in the nervous system. To test this assumption, organotypical rat hippocampal slice cultures were infected with BDV for up to 4 weeks. This resulted in the secretion of IFN and the up-regulation of IFN-stimulated genes. Using the rat Mx protein as a specific marker for IFN-induced gene expression, astrocytes and microglial cells were found to be Mx positive, whereas neurons, the major cell type in which BDV is replicating, lacked detectable levels of Mx protein. In uninfected cultures, neurons also remained Mx negative even after treatment with high concentrations of IFN-α. This non-responsiveness correlated with a lack of detectable nuclear translocation of both pSTAT1 and pSTAT2 in these cells. Consistently, neuronal dissemination of BDV was not prevented by treatment with IFN-α. These data suggest that the poor innate immune response in rat neurons renders this cell type highly susceptible to BDV infection even in the presence of exogenous IFN-α. Intriguingly, in contrast to rat neurons, IFN-α treatment of mouse neurons resulted in the up-regulation of Mx proteins and block of BDV replication, indicating species-specific differences in the type I IFN response of neurons between mice and rats.     

Modulation of C2 and C3 gene expression of human peripheral blood monocytes by interleukin 1β, interferon γ, tumor necrosis factor α and lipopolysaccharide

The effect of interleukin 1β (IL-1β), interferon γ (IFN-γ), tumor necrosis factor α (TNFα) and lipopolysaccharide (LPS) on the expression of the C2 and C3 genes in human adherent monocytes was studied. Stimulation of monocytes with IFN-γ increased both C2 and C3 mRNA. IL-1β also increased C2 mRNA level, whereas C3 gene expression was not enhanced. TNFα failed to increase either C2 or C3 mRNA. LPS increased C2 mRNA, but suppressed C3 gene expression. These results suggest that C2 and C3 production by monocytes is regulated by IL-1β and IFN-β in the local tissues.     

Early skin responses of hibernating and nonhibernating ground squirrels to topical applications of DMBA

Skin patches on hibernating and nonhibernating ground squirrels were treated with multiple topical applications of the carcinogen 7,12-dimethylbenza(a)anthracene. Nonhibernators showed blistering, peeling, drying, hair loss, increased vascularization and hyperpigmentation in proportion to DMBA concentration. The latter was apparently due to a) an increased number of dermal and epidermal melanocytes and b) the appearance of melanocytes with large coarse cytoplasmic granules. Notably, hibernators remained free of gross skin changes and were histologically similar to untreated controls.