Effects of low frequency pulsed electrical current on keratinocytes in vitro

The effects of low frequency pulsed electrical current on epidermal repair in vitro were examined. Charge-balanced current stimuli proposed for chronic wound treatment were tested on skin keratinocytes cultured at an air-liquid interface on dead human dermis. Results imply that the balance between proliferation and differentiation in electrically treated samples is significantly modified in favor of differentiation. More advanced differentiation, shown through epidermal histology, was obtained in cultures exposed to electrical current, whereas the culture growth, the result of keratinocyte migration and proliferation, was greater in control samples. Bioelectromagnetics 18:250–254, 1997. © 1997 Wiley-Liss, Inc.     

Decorin gene expression and its regulation in human keratinocytes

In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.     

Role of the Bullous Pemphigoid Antigen 180 (BP180) in the Assembly of Hemidesmosomes and Cell Adhesion—Reexpression of BP180 in Generalized Atrophic Benign Epidermolysis Bullosa Keratinocytes

Bullous pemphigoid antigen 180 (BP180) is a transmembrane component of hemidesmosomes (HD), cell–substrate attachment complexes in stratified and complex epithelia. To determine the role of BP180 in the assembly of HD and cell adhesion, using SV40 virions we have immortalized BP180-deficient keratinocytes derived from a patient with the inherited skin blistering disorder generalized atrophic benign epidermolysis bullosa (GABEB). The GABEB keratinocytes form HD-like structures, which contain α6β4 integrin and HD1/plectin, but not the bullous pemphigoid antigen 230 (BP230). The expression of integrin subunits by GABEB keratinocytes was comparable to that of an immortalized normal human keratinocyte cell line (NHK), except for α6 and β4, which were less strongly expressed in GABEB cells. In short-term adhesion assays, both GABEB keratinocytes and NHK bound strongly and to a similar extent to laminin-1, laminin-5, fibronectin, and type IV and V collagens, which suggests that BP180 is not involved in promoting the initial adhesion to these ligands. Transfection of GABEB keratinocytes with cDNAs for wild-type or a mutant of BP180 lacking the collagenous extracellular domain resulted in the expression of recombinant BP180 proteins that were correctly polarized at the basal cell surface together with α6β4. In addition, restored synthesis of BP180 affected the subcellular localization of BP230, which was no longer diffusely distributed in the cytoplasm, but was found in HD-like structures. In contrast, a BP180 mutant with a 36-amino-acid deletion from the amino terminus of the cytoplasmic domain failed to localize to HD-like structures. These results demonstrate that a region within the cytoplasmic domain of BP180 is essential for its localization into HD and that BP180 may play a critical role in coordinating the subcellular distribution of BP230.     

High-level secretion of growth hormone by retrovirally transduced primary human keratinocytes

A gene therapy clinical trial for treatment of growth hormone (GH) deficiency has not been reached yet, but several strategies using different gene transfer methodologies and animal models have been developed and showed successful results. We have set up an ex vivo gene therapy protocol using primary human keratinocytes transduced with an efficient retroviral vector (LXSN) encoding the human (hGH) or mouse GH (mGH) genes. These stably modified cells presented high in vitro expression levels of hGH (7 μg/10⁶ cells/d) and mGH (11 μg/10⁶ cells/d) after selection with geneticin. When the hGH-secreting keratinocytes were grafted onto immunodeficient dwarf mice (lit/scid), hGH levels in the circulation were about 0.2–0.3 ng/mL during a 12-d assay and these animals presented a significant body weight increase (p<0.01) compared to the control. Substitution of conventional grafting methodologies with organotypic raft cultures revealed a peak value of up to 20 ng mGH/mL in the circulation of grafted lit/scid mice at 1 h postimplantation, followed by a rapid decline to baseline (≈2 ng/mL) within 24 h. One week after grafting, however, the cultured excised implants still presented approx 45% of their original in vitro secretion efficiency. Further studies are being carrier out to identify the main factor(s) that still constitute one of the major impediments to the success of this promising model of cutaneous gene therapy.     

A ROCK inhibitor promotes keratinocyte survival and paracrine secretion,enhancing establishment of primary human melanocytes and melanocyte–keratinocyte co‐cultures

Primary melanocytes isolated from skin and expanded in culture have been widely used for laboratory research and clinical applications. The conventional method to isolate primary melanocytes from skin usually requires about 3–4 weeks of culture for melanocytes to grow sufficiently to passage. Considering that melanocytes comprise only 3%–7% of epidermal cells in normal human skin, it would be extremely helpful to increase the isolation efficiency and shorten the initial culture time to quickly meet various application needs. Here, we report that adding Y‐27632, a Rho kinase inhibitor, into the initial culture medium for 2 days can dramatically increase the yield of melanocytes. We found that Y‐27632 can promote keratinocyte attachment and survival in the melanocyte culture system, resulting in not only better recovery, but also increased proliferation of melanocytes by a paracrine signaling pathway. More specifically, Y‐27632 significantly induced keratinocyte expression of stem cell factor, which played an important role in enhancing the growth of melanocytes. In summary, Y‐27632 could profoundly enhance the yield of primary melanocytes in the initial culture through paracrine effects on keratinocytes.     

Adhesion of Human Epidermal Keratinocytes to Laminin

We have examined the mechanism by which human epidermal keratinocytes adhere to the A/B1/B2 (α₁β₁γ₁) form of laminin. Adhesion could be completely inhibited with an antibody to the β₁ integrin subunit or a combination of antibodies recognising the α₂β₂ a₃β₁ and α₆β₄ integrins. Keratinocytes adhered in the presence of magnesium and manganese ions, but calcium ions did not support adhesion and inhibited adhesion when combined with magnesium and manganese. The effects of anti-integrin antibodies (including a stimulatory antibody to the β₁ subunit) were not influenced by specific cations, with the exception that inhibition by an antibody to α₂β₁ was abrogated by the presence of manganese ions. The E3 and E8 proteolytic fragments of laminin did not support keratinocyte adhesion and heat inactivation of the E8 site in intact laminin did not reduce adhesion. Three laminin fragments that did support adhesion were P1, E4 and E1X-Nd, P1 activity being attributable at least in part to the RGD site; antibody blocking experiments suggested that adhesion to these fragments was primarily via α₁β₃. The synthetic peptide GD-6, derived from the carboxy terminus of the laminin A chain (included within E3) did support adhesion, but the significance of this observation is unclear, since a scrambled control peptide could also support adhesion. In conclusion, keratinocyte adhesion to A/B1/B2 laminin involves three integrins and multiple binding sites that are different from those defined previously.     

Mechanisms regulating epidermal stem cells

The skin epidermis contains different appendages such as the hair follicle and the sebaceous glands. Recent studies demonstrated that several types of stem cells (SCs) exist in different niches within the epidermis and maintain discrete epidermal compartments, but the exact contribution of each SC populations under physiological conditions is still unclear. In addition, the precise mechanisms controlling the balance between proliferation and differentiation of epidermal SC still remain elusive. Recent studies provide new insights into these important questions by showing the contribution of hair follicle SC to the sebaceous lineage and the importance of chromatin modifications and micro-RNAs (miRs) in regulating epidermal SCs renewal and differentiation. In this review, we will discuss the importance of these papers to our understanding of the mechanisms that control epidermal SC functions.     

Modulation of sulfur mustard induced cell death in human epidermal keratinocytes using IL-10 and TNF-alpha

We compared the effects of overexpressing a tightly regulated anti-inflammatory cytokine, interleukin 10 (IL-10), and the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on sulfur mustard induced cytotoxicity in human epidermal keratinocytes. Both cytokines were overexpressed when compared with the cells transfected with the empty vector as determined by quantitative ELISA. Cells overexpressing interleukin 10 suppressed the pro-inflammatory cytokines interleukin 8 and interleukin 6 following exposure to 50-300 microM sulfur mustard. These cells exhibited delayed onset of sulfur mustard induced cell death. On the other hand, cells overexpressing tumor necrosis factor alpha induced a sustained elevation in both interleukin 6 and 8 expression following exposure to 50-300 microM sulfur mustard. These cells were sensitized to the effects of sulfur mustard that resulted in an increased sulfur mustard induced cell death. Normal human epidermal keratinocytes treated with sulfur mustard exhibited elevated levels of tumor necrosis factor alpha expression and increased activity of nuclear factor kappa B. Gene array data indicated that cells overexpressing interleukin 10 induced several genes that are involved in growth promotion and cell-fate determination. We, therefore, identify IL-10 and TNF-alpha signal transduction pathways and their components as possible candidates for early therapeutic intervention against sulfur mustard induced cell injury.