Toru Fukaya

Substance P induces intracellular calcium increase and translocation of protein kinase C in epidermis

Substance P is a neuropeptide present in, and released from, peripheral C nerve endings. The presence of substance P‐positive nerve fibres in the epidermis has been reported. We investigated the effect of substance P on the transmembrane signalling system of pig epidermal keratinocytes. Treatment of pig epidermis with substance P resulted in an increase in inositol 1,4,5‐trisphosphate (IP3), and in intracellular free calcium. The treatment also resulted in translocation of protein kinase C from a cytosol to a membrane fraction. Substance P, however, did not affect the β‐adrenergic‐ or histamine (H2)‐ adenylate cyclase responses of the epidermis. Neither forskolin‐induced, nor cholera toxin‐induced cyclic AMP accumulation were affected by substance P treatment. These results are consistent with the view that substance P stimulates phosphatidylinositol‐4,5‐bisphosphate (PIP2) hydrolysis of keratinocytes, resulting in IP3‐Ca2+ and diacylglycerol‐protein kinase C signal activation. Although protein kinase C is known to affect the epidermal adenylate cyclase system, no evidence for such ‘cross‐talk regulation’ was detected in keratinocytes by substance P treatment.

Retinoid stimulates epidermal outgrowth of pig skin explants.

Using a pig explant culture system, the effects of retinoids on pig epidermal cells were studied. Ro 10‐9359 slightly stimulated epidermal outgrowth, but this effect was not significant. Ro 10‐1670 (a metabolite of Ro 10‐9359) significantly stimulated epidermal outgrowth. Cytochalasin B and colchicine markedly inhibited the stimulatory effect of Ro 10‐1670, but hydroxyurea or cycloheximide did not completely block this stimulatory effect. During a migratory period, cytochalasin B completely blocked it. Neither Ro 10‐1670 nor Ro 10‐9359 effected a change in mitotic index. Histological studies revealed that Ro 10‐1670‐treated epidermal cells did not form any keratin layers. These results suggest that Ro 10‐1670 stimulated outgrowth, particularly migratory outgrowth, of epidermal cells, resulting in reduced keratin formation.

Adenylate cyclase induces intracellular Ca2+ increase in single human epidermal keratinocytes of the epidermal sheet as measured by digital imaging microscopy using Fura 2-AM

SummaryIntracellular Ca2+ ([Ca2+]i) is thought to act as a second messenger of transmembrane signalling systems. However, no measurement of [Ca2+]i has been made in intact epidermal keratinocytes. We have developed a method for measuring [Ca2+]i in human keratinocytes from pure epidermal sheet by the application of digital imaging fluorescence microscopy with the use of Fura 2-AM. Normal human pure epidermal sheets were obtained by dispase treatment. Epinephrine and salbutamol induced transient [Ca2+]i increases. Propranolol, a Β-antagonist, inhibited this response, while prazosin and yohimbine (alpha1- and alpha2-antagonists, respectively) did not affect the response. Histamine and adenosine, also receptor agonists of the epidermal adenylate cyclase system, induced a similar [Ca2+]i increase, as did forskolin, a direct activator of adenylate cyclase. These data coincide with those previously presented for cultured human epidermal keratinocytes, and reveal that adenylate cyclase activation induces an increase of [Ca2+]i in intact epidermal cells. This technique enables the kinetics of [Ca2+]i in various skin disorders to be investigated.

Adenylate cyclase induces intracellular calcium increase in single human epidermal keratinocytes measured by fluorescence microscopy using Fura 2-AM.

Intracellular calcium ([Ca2+]i) is an important second messenger of extracellular signals to induce various cellular responses. Extracellular and intracellular Ca2+ are considered to be important for cellular differentiation and proliferation of epidermal keratinocytes. Several mechanisms which increase [Ca2+]i have been demonstrated in various tissues, but in epidermal keratinocytes these mechanisms are poorly understood. In epidermal keratinocytes the adenylate cyclase‐cyclic AMP response is thought to regulate cell proliferation and differentiation. However, the series of reactions which follow the cyclic AMP response remain unknown. Beta‐adrenergic agonists increase [Ca2+]i in cultured epidermal keratinocytes, and we have therefore studied whether stimulation of keratinocyte adenylate cyclase could induce [Ca2+]i increase, by using fluorescence microscopy with Fura 2‐AM.

Translocation of protein kinase C from cytosol to membrane fractions in human epidermal keratinocytes by recombinant human interferon-gamma

The activation of protein kinase C involves its translocation from a cytosol fraction to a membrane fraction. Effects of interferon-gamma (IFN-gamma) on the epidermal protein kinase C were investigated. The treatment of recombinant human IFN-gamma on intact human epidermis resulted in the translocation of protein kinase C from a cytosol to a membrane fraction. The human IFN-gamma had no translocation effect on pig epidermal protein kinase C. Tumor promoter, 12-o-tetradecanoylphorbol-13-acetate (TPA), and a membrane-permeable diacylglycerol analogue, 1-oleoyl-2-acetylglycerol (OAG), both of which are well-known activators of protein kinase C, translocated the epidermal protein kinase C. The IFN-gamma had no direct effect on the epidermal protein kinase C; the addition of the IFN-gamma to partially-purified pig epidermal protein kinase C had no effect on its activity. The effect of the IFN-gamma on human epidermal protein kinase C appears to be through the species specific IFN-gamma receptors. It has been reported that the epidermal beta-adrenergic adenylate cyclase response is decreased following the TPA- (and OAG-) induced activation of protein kinase C. Human recombinant IFN-gamma, however, had no effect on the beta-adrenergic response of the human epidermis. Our results indicate that IFN-gamma affects intact keratinocytes in vitro, resulting in the activation of protein kinase C, which might be related to the physiological effect of IFN-gamma on keratinocyte.

Triple Cancers in the Urogenital Area of a Patient with Aplastic Anemia

Three epithelial neoplastic lesions, perineal Bowenoid papulosis, uterine cervical carcinoma, and bladder transitional cell carcinoma, which occurred in a mildly immunosuppressed patient who had aplastic anemia were studied for human papillomavirus (HPV) infection. In the Bowenoid papulosis, HPV type 16 DNA was identified by polymerase chain reaction (PCR) and by in situ hybridization (ISH). In contrast, in the uterine cervical carcinoma, HPV 16 was not detected, although possibly another unidentified type of HPV in the lesion was suggested by the ISH findings. In the bladder transitional cell carcinoma, neither papillomavirus genus‐specific (PGS) antigen nor HPV DNA was found.

Adult T cell leukemia: A case with massive hyperimmunoglobulinemia E

Adult T cell leukemia, an endemic disease in the southwestern part of Japan, is characterized (1) by a short survival, (2) by leukemic T cells in peripheral blood that have lobulated nuclei and helper/inducer surface phenotypes, and (3) by cutaneous involvement. A 34-year-old man who had a history of atopic dermatitis was seen at our clinic because of generalized erythroderma and lymphadenopathy. His clinical course was rather chronic as compared with that of prototypic adult T cell leukemia; however, typical leukemia cells were observed in specimens taken from his peripheral blood and skin. The diagnosis of adult T cell leukemia was established by the patients positive serum antibody reaction to adult T cell leukemia-associated antigen and monoclonal integration of virus genome in the patients leukemia cell DNA. Interesting and characteristic of the patient were the very high levels of serum immunoglobulin E. With the use of an in vitro immunoglobulin production system with mitogen, the patients T lymphocytes enhanced the differentiation of B cells, both from the patient and from a normal adult, into immunoglobulin E-producing cells. Therefore it may be speculated that T cells functioning as immunoglobulin E-specific helpers were transformed to leukemia cells by human T-lymphotropic virus type I. Continuous antigen stimulation of the patients atopic diathesis also may be a factor.

Effects of calcium and calcium-ionophore on the outgrowing epidermis--possible activation of epidermal phospholipase A2.

Using an in vitro explant culture system of pig skin, the effects of calcium and calcium‐ionophore on the outgrowing epidermis were studied. The optimum concentration of calcium on the rates of epidermal outgrowth was 1.0 mM and that of mitosis was around 1.2 mM. Ionophore A23187 (Ionophore) significantly stimulated both the rates of epidermal outgrowth and the mitosis of the outgrowing epidermis. This stimulation was partially blocked by the addition of hydrocortisone.

VARIOUS CELLULAR ACTIVITIES OF THE OUTGROWING EPIDERMIS IN PIG SKIN EXPLANT CULTURE—THE EFFECTS OF METABOLIC INHIBITORS

The effects of metabolic inhibitors on the outgrowing epidermis were studied to detect the various cellular activities of epidermal cells. Hydroxyurea significantly inhibited mitosis and 3H‐thymidine uptake, but the inhibitory effect of hydroxyurea on the rate of epidermal outgrowth was less remarkable. On the other hand, cytochalasin B significantly inhibited the rate of epidermal outgrowth but did not inhibit mitosis or 3H‐thymidine uptake. The overall results indicate that there are several cellular activities such as migration and proliferation in the outgrowing epidermis in vitro.