Clare Gordon-Thomson

Evidence for a Specific Uptake and retention Mechanism for 25- Hydroxyvitamin D (25OHD) in skeletal Muscle Cells

Little is known about the mechanism for the prolonged residence time of 25-hydroxyvitamin D (25OHD) in blood. Several lines of evidence led us to propose that skeletal muscle could function as the site of an extravascular pool of 25OHD. In vitro studies investigated the capacity of differentiated C2 murine muscle cells to take up and release 25OHD, in comparison with other cell types and the involvement of the membrane protein megalin in these mechanisms. When C2 cells are differentiated into myotubes, the time-dependent uptake of labeled 25OHD is 2-3 times higher than in undifferentiated myoblasts or nonmuscle osteoblastic MG63 cells (P < .001). During in vitro release experiments (after 25OHD uptake), myotubes released only 32% ± 6% stored 25OHD after 4 hours, whereas this figure was 60% ± 2% for osteoblasts (P < .01). Using immunofluorescence, C2 myotubes and primary rat muscle fibers were, for the first time, shown to express megalin and cubilin, endocytotic receptors for the vitamin D binding protein (DBP), which binds nearly all 25OHD in the blood. DBP has a high affinity for actin in skeletal muscle. A time-dependent uptake of Alexafluor-488-labeled DBP into mature muscle cells was observed by confocal microscopy. Incubation of C2 myotubes (for 24 hours) with receptor-associated protein, a megalin inhibitor, led to a 40% decrease in 25OHD uptake (P < .01). These data support the proposal that 25OHD, after uptake into mature muscle cells, is held there by DBP, which has been internalized via membrane megalin and is retained by binding to actin.

Photoprotection by 1α,25-dihydroxyvitamin D and analogs: Further studies on mechanisms and implications for UV-damage

Ultraviolet (UV) irradiation causes DNA damage in skin cells, immunosuppression and photocarcinogenesis. 1alpha,25-dihydroxyvitamin D3 (1,25D) reduces UV-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in human keratinocytes in culture and in mouse and human skin. UV-induced immunosuppression is also reduced in mice by 1,25D, in part due to the reduction in CPD and a reduction in interleukin (IL-6. The cis-locked analog, 1alpha,25-dihydroxylumisterol3 (JN), which has almost no transactivating activity, reduces UV-induced DNA damage, apoptosis and immunosuppression with similar potency to 1,25D, consistent with a non-genomic signalling mechanism. The mechanism of the reduction in DNA damage in the form of CPD is unclear. 1,25D doubles nuclear expression of p53 compared to UV alone, which suggests that 1,25D facilitates DNA repair. Yet expression of a key DNA repair gene, XPG is not affected by 1,25D. Chemical production of CPD has been described. Incubation of keratinocytes with a nitric oxide donor, SNP, induces CPD in the dark. We previously reported that 1,25D reduced UV-induced nitrite in keratinocytes, similar to aminoguanidine, an inhibitor of nitric oxide synthase. A reduction in reactive nitrogen species has been shown to facilitate DNA repair, but in view of these findings may also reduce CPD formation via a novel mechanism.

ErbB receptors mediate both migratory and proliferative activities in human melanocytes and melanoma cells

Epidermal growth factor receptor (EGFR) activation by transforming growth factor alpha (TGF&agr;) has been implicated in autocrine growth in melanoma, but does not alter melanocyte proliferation. This raises the possibility that different signalling pathways are activated via EGFR or ErbB receptors. Here, we demonstrate that ErbB2, ErbB3 and ErbB4 are expressed in cultured human melanocytes. Western analyses with receptor-specific antisera revealed protein bands with Mr values of 185 and 160 kDa, corresponding to ErbB2 and ErbB3, respectively. Blots probed with ErbB4 antibodies showed bands with Mr values of 180, 120 and 80 kDa, corresponding to the receptor and its reported variants. Two malignant melanoma cell lines expressed ErbB2 and ErbB3, but not the full-length ErbB4 receptor. As TGF&agr; binds to EGFR and the heregulins (HRG) bind to ErbB3 and ErbB4, these growth factors were examined for effects on receptor activation and on cell growth and motility in a scratch wound closure assay. In normal melanocytes, HRG&bgr;1 activated the phosphorylation of tyrosine residues of proteins that immunoprecipitated with EGFR and ErbB4 antisera, and significantly enhanced cell migration but not proliferation. Neither TGF&agr; nor HRG&agr;1 promoted migration or growth in normal melanocytes. By contrast, TGF&agr; stimulated migratory activity in the MM96L cell line, but not in the MELJG line, whereas HRG&bgr;1 significantly enhanced cell growth, but not migration, in both malignant cell lines. The apparent transition of HRG&bgr;1 from a migratory to a proliferative function after malignant transformation, and the change in TGF&agr; from a non-migratory to a migratory activity in one melanoma line, suggests multiple switches in ErbB signalling pathways via EGFR/ErbB heterodimer formation.

Vitamin D: the light side of sunshine

Under normal circumstances, vitamin D is mainly obtained from skin through the action of ultraviolet B irradiation on 7-dehydrocholesterol. It is further metabolized to 25-hydroxyvitamin D (25OHD), the major circulating vitamin D compound, and then to 1,25-dihydroxyvitamin D, the hormonal form. The major function of vitamin D compounds is to enhance active absorption of ingested calcium (and phosphate). This assists in building bone at younger ages and ensures that despite obligatory urinary losses, bone does not need to be resorbed to maintain blood calcium concentrations. Vitamin D compounds appear to have direct effects to improve bone and muscle function, and there is good, although not entirely consistent, evidence that supplemental vitamin D and calcium together reduce falls and fractures in older individuals. On the basis of calcium control and musculoskeletal function, target levels for 25OHD in blood are at least 50–60 nmol/l and there may be a case for higher targets of 75–80 nmol/l. There are vitamin D receptors in most nucleated cells and some evidence, although not consistent, that adequate vitamin D levels may be important in reducing the incidence of, or mortality from, some cancers and in reducing autoimmune disease. Adequate vitamin D may also allow for a normal innate immune response to pathogens, improve cardiovascular function and mortality and increase insulin responsiveness. Vitamin D levels are maintained better in the presence of adequate calcium intakes, more exercise and less obesity. Genetic variation may have an effect on vitamin D blood levels and response to treatment with vitamin D.

The Role of the Vitamin D Receptor and ERp57 in Photoprotection by 1α,25-Dihydroxyvitamin D3

UV radiation (UVR) is essential for formation of vitamin D(3), which can be hydroxylated locally in the skin to 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. Recent studies implicate 1,25-(OH)(2)D(3) in reduction of UVR-induced DNA damage, particularly thymine dimers. There is evidence that photoprotection occurs through the steroid nongenomic pathway for 1,25-(OH)(2)D(3) action. In the current study, we tested the involvement of the classical vitamin D receptor (VDR) and the endoplasmic reticulum stress protein 57 (ERp57), in the mechanisms of photoprotection. The protective effects of 1,25-(OH)(2)D(3) against thymine dimers were abolished in fibroblasts from patients with hereditary vitamin D-resistant rickets that expressed no VDR protein, indicating that the VDR is essential for photoprotection. Photoprotection remained in hereditary vitamin D-resistant rickets fibroblasts expressing a VDR with a defective DNA-binding domain or a mutation in helix H1 of the classical ligand-binding domain, both defects resulting in a failure to mediate genomic responses, implicating nongenomic responses for photoprotection. Ab099, a neutralizing antibody to ERp57, and ERp57 small interfering RNA completely blocked protection against thymine dimers in normal fibroblasts. Co-IP studies showed that the VDR and ERp57 interact in nonnuclear extracts of fibroblasts. 1,25-(OH)(2)D(3) up-regulated expression of the tumor suppressor p53 in normal fibroblasts. This up-regulation of p53, however, was observed in all mutant fibroblasts, including those with no VDR, and with Ab099; therefore, VDR and ERp57 are not essential for p53 regulation. The data implicate the VDR and ERp57 as critical components for actions of 1,25-(OH)(2)D(3) against DNA damage, but the VDR does not require normal DNA binding or classical ligand binding to mediate photoprotection.

Regulation of epidermal growth factor receptor expression in human melanocytes.

Abstract: The epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor alpha (TGFα), are reportedly involved in autocrine growth of melanoma cells. The signal pathway has also been implicated in early events of transformation, suggesting a function for EGFR in normal cells. This study reports the presence of EGFR in cultured melanocytes and examines some cellular responses to TGFα. Western analysis revealed 170 kDa bands in extracts of cultured neonatal human melanocytes, corresponding to the receptor Mr. Protein expression was more pronounced in cells during active growth. EGFR were less evident in cultures populated predominantly by melanized cells, indicating that receptor expression became reduced in differentiating cells. Immunocytochemistry confirmed these observations and also showed that EGFR reactivity was predominantly localized in the cell body but absent from dendrites. Addition of TGFα to early cultures induced a rapid increase in phosphotyrosine signal of the 170 kDa protein. Longer treatment (24–48 h) increased the intensity of the EGFR signal, suggesting that receptors had been upregulated. However, inclusion of TGFα in cultures did not result in an increase in cell numbers when compared to controls. The observations provide evidence of the existence of a receptor‐mediated pathway in melanocytes which has transforming potential in vivo.

Chitotriosidase and gene therapy for fungal infections

Abstract.Chitotriosidase secreted by activated human macrophages has been implicated in the defence against chitin-bearing pathogens. The antifungal properties of human chitotriosidase were investigated here following retroviral vector-mediated gene transfer of the open reading frame of the chitotriosidase gene into Chinese hamster ovary cells. A chitinase assay confirmed that the engineered cells secreted recombinant chitotriosidase constitutively. Two dimensional gel electrophoresis and western blotting indicated that the recombinant protein is the major, chitin-binding, fifty kilodalton isoform. Culture medium conditioned by the transduced cells inhibited growth of isolates of Aspergillus niger, Candida albicans and Cryptococcus neoformans. Furthermore, longevity was significantly increased in a mouse model of cryptococcosis when cells transduced with the chitotriosidase gene and encapsulated in alginate microspheres were implanted subcutaneously in the animals. Engraftment of microcapsules containing cells transduced with the chitotriosidase gene has the potential to combat infections caused by chitinous pathogens through the prolonged delivery of recombinant chitotriosidase.

Vitamin D and Death by Sunshine

Exposure to sunlight is the major cause of skin cancer. Ultraviolet radiation (UV) from the sun causes damage to DNA by direct absorption and can cause skin cell death. UV also causes production of reactive oxygen species that may interact with DNA to indirectly cause oxidative DNA damage. UV increases accumulation of p53 in skin cells, which upregulates repair genes but promotes death of irreparably damaged cells. A benefit of sunlight is vitamin D, which is formed following exposure of 7-dehydrocholesterol in skin cells to UV. The relatively inert vitamin D is metabolized to various biologically active compounds, including 1,25-dihydroxyvitamin D3. Therapeutic use of vitamin D compounds has proven beneficial in several cancer types, but more recently these compounds have been shown to prevent UV-induced cell death and DNA damage in human skin cells. Here, we discuss the effects of vitamin D compounds in skin cells that have been exposed to UV. Specifically, we examine the various signaling pathways involved in the vitamin D-induced protection of skin cells from UV.

A novel model of wound healing in the SCID mouse using a cultured human skin substitute

Studies of skin graft behaviour in rodent excisional wound models are limited by the dominance of wound contracture and graft sloughing as primary healing responses. To slow skin contraction, polytetrafluoroethylene (Teflon) rings were inserted into dorso‐lateral full‐thickness wounds in SCID mice. Cultured skin substitutes (OrCel), composed of cultured human keratinocytes and fibroblasts in a bovine collagen sponge, were implanted within the rings. Examination and histology of grafts 14 days later showed graft take in four of six recipients, with 90% epithelialization and wound contraction of 31–47%. Immunohistochemical studies, using human‐specific antisera to distinguish graft from host tissues, showed that regenerated tissue was predominantly human. Staining with anticytokeratin, revealed a multilayered, stratified neoepidermis. HBG were identified in keratinocytes in all epidermal layers. Langerhans cells were absent. Antihuman vimentin, used as a fibroblast marker, confirmed that cells of the neodermis were primarily of human origin. Neoepidermal keratinocytes, primarily in the basal and suprabasal layers, were also stained. Results suggest that the poly(tetrafluoroethylene) ring inhibited graft sloughing and provided a more favourable environment for the skin substitute to regenerate a substantially normal human skin.

Parathyroid hormone-related peptide modulates signal pathways in skin and hair follicle cells.

Abstract:  Parathyroid hormone‐related protein (PTHrP) is secreted by skin epithelial cells and is thought to play an important role in the development and function of the hair follicle. It was hypothesized that PTHrP binds to receptors in dermal papilla cells and modulates intracellular signaling systems in these cells. We tested the effects of PTHrP on protein synthesis, protein kinase A (PKA) and protein kinase C (PKC) activities as well as tyrosine phosphorylation in rat vibrissa dermal papilla and capsular fibroblast cells. Cells were cultured in the presence or absence of the N‐terminal peptide PTHrP1‐34 for 48 h and detergent extracts prepared. Proteins were separated by electrophoresis. Phosphotyrosine and the PTH/PTHrP receptor immunoreactivity was identified by Western blot analysis. PKC and PKA activities in the cells were measured using colorimetric enzyme assays. Extracts of both dermal papilla cells and capsular fibroblasts displayed immunoreactivity to the PTH/PTHrP receptor. Electrophoresis showed that PTHrP treatment reduced the density of a 50‐kDa protein in dermal papilla cells but not in capsular fibroblasts. Media conditioned by the cells showed similar changes, indicating that the PTHrP‐modulated 50‐kDa protein was secreted. Furthermore, 2‐D gel electrophoresis indicated that the protein had a number of phosphorylation sites. Western analysis with antiphosphotyrosine antibodies confirmed a significant decrease in the intensity of a phosphorylated 50‐kDa protein in papilla cells and papilla cell‐conditioned medium. PKC and PKA activities of papilla cells were unaffected by PTHrP. However, activities of PKC were increased and PKA reduced in capsular fibroblasts following peptide treatment. These cell‐specific effects showed that endogenous PTHrP may activate different intracellular pathways in mesenchymal cells of skin and elicit changes in levels of locally secreted proteins that specifically modulate normal follicular function.