ADP-ribosylation of the rhoA gene product by botulinum C3 exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of the cell cycle.

Using botulinum C3 exoenzyme, which specifically ADP-ribosylates the rho gene products (rho proteins), we examined the role of these proteins in cell cycle progression in Swiss 3T3 cells. Incubation of cell lysates with C3 exoenzyme revealed a single [32P]ADP-ribosylated protein with an M(r) of 23K. This protein was identified as rhoA protein by isoelectric focusing and peptide mapping. When C3 exoenzyme was added to the culture, it ADP-ribosylated the substrate protein in the cells and reduced their growth rate and saturation density. The reduction was dependent on the amount of C3 exoenzyme and on the extent of ADP-ribosylation of the rho protein in the cells. Flow cytometric analysis of logarithmically growing cells showed that the enzyme treatment concentration-dependently accumulated the cells in the G1 phase of the cell cycle. When G1-enriched cells were treated with C3 exoenzyme and cell cycle progression initiated by the addition of serum was monitored, inhibition of G1-S transition was clearly observed. These results suggest that the rhoA gene product plays a critical role in G1-S progression in cultured Swiss 3T3 cells and that the ADP-ribosylation abolishes this activity and causes the cells to accumulate in G1 phase.

Phosphorylation of keratin polypeptides.

The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing 32Pi, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weights between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [gamma-32P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [gamma-32P]ATP and cyclic AMP-dependent protein kinase from calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation.

Leukotriene A4 hydrolase in human skin.

The biochemical properties and immunohistochemical localization of leukotriene (LT) A4 hydrolase were investigated in human skin. The activity of LTA4 hydrolase, which catalyzes the hydrolysis of LTA4 to LTB4, the most chemotactic compound known, was detected in the 100,000 x g supernatant of homogenates of human epidermis and a transformed epidermal cell line (HSC-1). No significant LTA4 hydrolase activity was detected in human whole skin or dermis. The enzymatic properties of LTA4 hydrolase isolated from human keratinocytes and peripheral leukocytes were similar. Their activities were inhibited by bestatin and captopril, and they were completely absorbed by anti-human LTA4 hydrolase antibody. By immunoblotting analysis using this antibody, LTA4 hydrolase was detected as a 70-kDa protein in human epidermis and HSC-1 and was found to be similar to the enzyme detected in peripheral mononuclear leukocytes. In human dermis, LTA4 hydrolase was barely detected by Western blotting. On the other hand, LTA4 hydrolase was demonstrated in the cytoplasm of keratinocytes in the epidermis, and in fibroblasts, infiltrating and endothelial cells in the dermis of normal human skin by immunohistochemical analysis using the immunoperoxidase method. These results suggest that LTB4 can be generated from LTA4 by LTA4 hydrolase in keratinocytes as well as fibroblasts, infiltrating and endothelial cells in the dermis of human skin.

Induction of 72-kD heat shock protein and cytoskeleton damage by cytotoxic prostaglandin delta 12-PGJ2 in transformed human epidermal cells in culture.

Cyclopentenone prostaglandins (PG) such as delta 12-PGJ2 and PGA are potent inhibitors of growth in a variety of cultured cells, including human epidermal cells. To clarify the mechanism of PG cytotoxicity in human epidermal cells, we examined the effects of delta 12-PGJ2 on the induction of a heat shock protein (HSP), and on the organization of cytoskeletons in the HSC-I-transformed human epidermal cell line. Immunoblot analysis using a monoclonal antibody specific for the 72-kD heat shock protein (HSP72) revealed that a 12-h incubation with 5 micrograms/ml of delta 12-PGJ2 induced HSP72 formation in HSC-I cells. HSP72 was also induced by heat shock treatment at 43 degrees C for 90 min. The quantity of HSP72 produced was markedly decreased by co-treatment with 1 microgram/ml of cycloheximide in delta 12-PGJ2-treated cells, and similarly reduced in HSC-I cells following heat treatment. Immunofluorescence using a monoclonal antibody to HSP72 demonstrated that HSP72 was localized mainly in the cytoplasm of HSC-I cells. Following treatment with 5 micrograms/ml of delta 12-PGJ2, however, HSP72 was found in the nucleolus as well as in the cytoplasm. The accumulation of HSP in the nucleolus was similarly prominent in HSC-I cells after treatment at 43 degrees C for 90 min. Addition of delta 12-PGJ2 to confluent HSC-1 cells resulted in the disappearance of actin filaments and the disarrangement of keratin filaments, as visualized with fluorescent-labeled phallacidine or immunofluorescence. These results suggest that the cytotoxicity of cyclopentenone PG is related to the induction of HSP72, and to cytoskeleton damage in transformed human epidermal cells in culture.

Effect of ultraviolet irradiation on the activity of rat skin prostaglandin D synthetase.

The effect of ultraviolet light-B (UVB) irradiation on the activity of prostaglandin (PG) D synthetase was investigated in adult rat skin. Rats were irradiated with 500 mJ/cm2 of UVB, and PGD synthetase activity was determined in 100,000 g supernatant of the homogenate of rat skin in the presence of glutathione (GSH) before and 3, 6, 12, and 24 h after irradiation. The PGD synthetase activity was decreased time dependently, and within 24 h after UVB irradiation it had dropped to 50% of the control level before irradiation. In contrast, the synthesizing activities of PGE2 and PGF2 alpha were unaffected by UVB irradiation. The reduction of PGD synthetase activity after UVB irradiation was much more prominent in the epidermis than in the dermis, which was separated by heat treatment (55 degrees C, 30 sec). Immunohistochemical studies, using anti-(rat spleen PGD synthetase) antibody, revealed that the number of immunopositive cells, which were identified as Langerhans cells, decreased in the basal layer of the epidermis 24 h after UVB irradiation. These results, together with the reduction of ATPase positive cells in the epidermis after UVB irradiation, suggest that the decrease of PGD synthetase activity in rat skin by UVB irradiation is, at least in part, due to the reduced Langerhans cell population in the basal layer of the epidermis.

Effects of substance P and substance K on the growth of cultured keratinocytes.

The effects of substance P and substance K, which are coexpressed in the same mRNA as a beta-preprotachykinin in peripheral tissues and released in the inflammatory lesion of the skin, were examined on epidermal proliferation using spontaneously transformed mouse epidermal cell line (Pam 212 cells). Substance P stimulated the synthesis of DNA of Pam 212 cells in the medium containing 2%-10% fetal calf serum (FCS). Stimulation of DNA synthesis was dose dependent if the cells were cultured in the medium containing 2% FCS (quiescent condition). This effect was inhibited by spantide. In a serum-free medium, substance P had no effect on keratinocyte proliferation. In contrast, substance K, which shares a common amino acid sequence with substance P on its C-terminal, did not affect DNA synthesis of Pam 212 cells in either medium condition. Substance P released in inflammation may stimulate epidermal proliferation.

Characterization and distribution of prostaglandin D synthetase in rat skin.

The biochemical properties and immunohistochemical localization of prostaglandin D synthetase were investigated in adult rat skin. The activity of prostaglandin D synthetase, which isomerizes prostaglandin H2 to prostaglandin D2, was detected in the 100,000 g supernatant of the homogenate of adult rat skin. Whole skin showed considerable activity (1.9 nmol/min/mg protein), and prostaglandin D2 was the major prostaglandin among those formed from prostaglandin H2 in the presence of glutathione. The epidermis, which was separated from whole skin by heating (55 degrees C, 30 s), exhibited about three times higher activity (3.5) than the dermis (1.0). The enzymatic properties of both layers were similar; they were absolutely glutathione-dependent, were inhibited only a few percent by 1 mM 1-chloro-2,4-dinitro-benzene, and were completely absorbed by anti-rat spleen prostaglandin D synthetase antibody. Immunohistochemical studies, using anti-rat spleen prostaglandin D synthetase antibody and the immunoperoxidase method, showed that prostaglandin D synthetase was localized in Langerhans cells (not in keratinocytes) in the epidermis, in macrophages or histiocytes, and also in mast cells in the dermis. Immunoelectron microscopy also supported these findings. These results suggest that prostaglandin D2 is one of the most important arachidonic acid metabolites and plays a significant role in immunological function in the skin via Langerhans cells and macrophages.

Inhibition of the proliferation of transformed epidermal cells in culture by various prostaglandins.

Cytotoxic action of various prostaglandins (PGs) was examined on the PAM 212 transformed mouse epidermal cell line, and delta 7-PGA1 was found most active. delta 7-PGA1 exerted a dose-dependent inhibition of PAM 212 cell growth over 0.1 microgram/ml (0.3 microM). At 1.6 microgram/ml (4.6 microM) growth was completely inhibited, and the number of viable cells decreased remarkably during culture. The concentration needed for 50% growth inhibition (IC50) value of delta 7-PGA1 on PAM 212 cell growth was calculated as 0.4 microgram/ml (1.1 microM). At this concentration, the DNA synthesis in 24- and 48-h cultured cells was decreased to a half of the level in the control cells, and microscopically, remaining cells showed degenerative changes with many vacuoles in their cytoplasm. Prostaglandin D2, a major PG in mast cells, also showed potent cytotoxic activity. However, this action was expressed as 9-deoxy-delta 9,12-13,14-dihydro-PGD2 (delta 12-PGJ2), which was converted from PGD2 in plasma, and had a 3-fold stronger growth inhibitory activity than PGD2; the IC50 values of PGD2 and delta 12-PGJ2 were 2 micrograms/ml (5.7 microM) and 0.75 microgram/ml (2.1 microM), respectively. Among other PGs tested, PGA2 showed a comparable growth inhibitory activity, and PGB2, PGE1, and PGE2 less but significant activity. Prostaglandin F2 alpha and PGI2 however, had no such effect on cell proliferation at 5 micrograms/ml (14.3 microM) concentration, suggesting that cyclopentenone structure is an essential moiety of PG derivatives for cell growth inhibition. This cytotoxic action of delta 7-PGA1 and delta 12-PGJ2 appears to be independent of cyclic-AMP, since these PGs were virtually inactive in raising intracellular cyclic-AMP levels in PAM 212 cells.

Effect of ultraviolet irradiation on mast cell-deficient W/Wv mice.

The effect of UV irradiation on the skin was investigated in (WB-W/+) X (C57BL/6J-Wv/+)F1-W/Wv mice, which are genetically deficient in tissue mast cells. Their congenic littermates (+/+) and normal albino mice (ICR or BALB/c) were used as controls. Mice were irradiated with 500 mJ/cm2 of UVB and the increment of ear thickness was measured before and 6, 12, and 24 h after irradiation. Ear swelling in W/Wv mice at 12 and 24 h after irradiation was significantly smaller than that in +/+ and ICR mice. In contrast, the number of sunburn cells formed 24 h after UVB irradiation (200 or 500 mJ/cm2) was similar in W/Wv, +/+ and ICR mice. On the other hand, when mice were treated with 8-methoxy-psoralen (0.5%) plus UVA irradiation (4 J/cm2) (topical PUVA), ears of W/Wv and BALB/c mice, which were both white in color, were thickened similarly 72 h after treatment, but less swelling was observed in +/+ mice, which were black in skin color. The amount of prostaglandin D2 (PGD2) in ears, determined by radioimmunoassay specific for PGD2, was elevated 3-fold in +/+ and ICR mice at 3 h after irradiation with 500 mJ/cm2 of UVB in comparison with basal level without irradiation. However, such elevation was not observed in W/Wv mice. These results suggest that mast cells play an important role in UVB-induced inflammation, and PGs from mast cells are responsible at least in part for the development of this reaction. However, neither mast cells nor PGs contribute to the sunburn cell formation and ear swelling response by PUVA treatment.

Relationship between heat shock protein induction and the binding of antibodies to the extractable nuclear antigens on cultured human keratinocytes.

The importance of environmental factors such as ultraviolet light and temperature in the pathogenesis of cutaneous lupus erythematosus is well recognized. Recent evidence suggests the presence of autoantibodies to heat shock proteins (HSP) in the sera and enhanced expression of the HSP70 gene in peripheral blood mononuclear cells of patients with systemic lupus erythematosus. We designed experiments to determine how HSP or stress protein inducers affect the cell surface binding of IgG antibodies from sera containing anti-SS-A/Ro and anti-ribonuclear protein (RNP) antibodies to keratinocytes because these antibodies are considered to be one of the immunologic triggers of cutaneous lupus erythematosus. Immunofluorescence and immunoblot analysis using a monoclonal antibody to the 72 kDa of HSP revealed that an 18-h incubation with 10 micrograms/ml of delta 12-PGJ2, one of cytotoxic prostaglandins, induced HSP72 formation in cultured human keratinocytes. delta 12-PGJ2 augmented the binding of IgG antibodies from sera containing anti-U1RNP and anti-SS-A/Ro antibodies to cultured keratinocytes, but produced no enhancement of the binding of IgG antibodies from sera containing anti-Sm or anti-DNA antibodies. Similar results were also obtained by using flow cytometry analysis. HSP was also induced by ultraviolet B irradiation. These results suggest that exposure of keratinocytes to stressors such as delta 12-PGJ2 and ultraviolet light increases the binding sites for U1RNP,SS-A/Ro, and SS-B/La antibodies. The association between HSP induction and the appearance of extractable nuclear antigens may provide a better understanding of why environmental stimuli can promote the development of erythematous lesions in the skin.

A human T cell line established from a patient with Sézary syndrome. Application for assay of human interleukin 2 (IL-2).

A human T cell line, designated Sez 627, was established from a patient with Sézary syndrome. These clonal T cells have been cultured for more than 2 years in the presence of IL-2 without any antigen stimulation. The surface phenotype of Sez 627 was OKT3+, OKT4-, OKT8+, and Tac+, and infection with human T lymphotrophic virus type I (HTLV-I) was demonstrated by Southern blot hybridization analysis. In human IL-2 assay, Sez 627 cells were found to be superior to murine CTLL-2 cells with respect to their unresponsiveness to phorbol 12-myristate 13-acetate (PMA), and species specificity for human IL-2, as well as better cryopreservation, although they were less sensitive to IL-2 than CTLL-2 cells. Using Sez 627 cells, IL-2 production by lymphocytes from patients with systemic lupus erythematosus (SLE) was examined. Decreased IL-2 production was observed in the patients with active SLE but not in most patients with inactive SLE. These findings suggest that Sez 627 is a useful human T cell line for human IL-2 assay.

Immunohistochemical demonstration of poly(adenosine diphosphate-ribose) synthetase in bovine tissues.

The inter- and intracellular localization of poly(adenosine diphosphate-ribose)(poly(ADP-ribose] synthetase was investigated using an indirect immunofluorescence technique and a specific antibody against the enzyme purified from calf thymus. In various bovine tissues, including liver, heart, pancrease, thyroid, spleen, adrenal, and skeletal muscle, the specific immunofluorescence of poly(ADP-ribose) synthetase was localized exclusively in the nucleus. Immunostaining was inhibited by preabsorption of the antibody with purified calf thymus poly(ADP-ribose) synthetase. Nuclear immunofluorescence appeared to be more prominent in the marginal area than in the central region in most nuclei. This staining pattern is similar to that of naturally occurring poly(ADP-ribose). In bovine peripheral blood the immunofluorescence of poly(ADP-ribose) synthetase was detected in nuclei of lymphocytes, but not in granulocytes, in agreement with the finding that the enzymatic activity of poly(ADP-ribose) synthetase was barely detectable in nuclei isolated from granulocytes.

Immunohistochemical demonstration of poly(adenosine diphosphate-ribose) in nuclei of various rat tissues.

Natural distribution of poly(adenosine diphosphate-ribose), a novel macromolecule in eukaryotes, was investigated using an indirect immunofluorescence technique. The antibody, produced in a rabbit toward poly(ADP-ribose), was most reactive with polymers having the chain length of about 25 ADP-ribose units and weakly reactive with short oligomers; it was totally inert with monomers. Immunostaining with this antibody revealed the existence of the polymer in various rat tissues. The immunostaining seems to be specific for poly(ADP-ribose), as judged by its disappearance by preabsorption of the antiserum with purified poly(ADP-ribose) or pretreatment of tissue sections with poly(ADP-ribose)-degrading enzymes. Intensification of the fluorescence by preincubation with nicotinamide adenine dinucleotide (NAD), a substrate for poly(ADP-ribose) synthesis, also supported this view. The immunofluorescence of poly(ADP-ribose) was found exclusively in the nucleus of almost all tissues tested, including liver (adult, newborn, regenerating, and hepatoma), brain, heart, intestine, pancreas, kidney, spleen, testis, thyroid gland, and skeletal muscle. Exceptions were blood cells; little fluorescence was detectable in nuclei of peripheral leukocytes. Only after preincubation with NAD, did lymphocytes and monocytes exhibit fluorescence, however, granulocytes never did exhibit fluorescence. The cells appeared to represent the first instance where poly(ADP-ribose) synthesis activity among eukaryotic cells was missing.

Poly(ADP-ribose) synthesis in human cervical cancer cell-diagnostic cytological usefulness.

Poly(ADP-ribose) synthetase activity in human cervical cancer cell nuclei was found to be approximately twice that of normal cervical cell nuclei. Using antipoly(ADP-ribose) antibody, frozen section of normal tissue were stained by indirect-immunofluorescence and it was demonstrated that the fluorescence of nuclei in squamous cells decreased during maturation. Correspondingly, superficial cells appearing in smear specimens had less nuclear fluorescence compared with parabasal cells in the same smear. However, cancer cells in cervical smears had marked nuclear fluorescence using immunostaining and were easily distinguished from normal cells.

Inhibitory effect of tranilast on prostaglandin D synthetase.

The effect of Tranilast [N-(3,4-dimethoxycinnamoyl) anthranilic acid] on the synthesis of prostaglandin D2 (PGD2) by homogenates of rat peritoneal mast cells was investigated. The major cyclooxygenase product formed by mast cell homogenates was PGD2, smaller quantities of PGE2 and PGF2 alpha were also formed. Tranilast suppressed the production of PGD2 in a dose-dependent manner with an IC50 of 0.1 mM. This suppression was due to inhibition of PGD synthetase, but not cyclooxygenase, since the formation of PGE2 and PGF2 alpha were unchanged at a 0.1 mM concentration. In addition, the glutathione-dependent conversion of [14C]PGH2 to PGD2 by PGD synthetase (PGH-D isomerase, EC 5.3.99.2) was inhibited by Tranilast, with 50% inhibition achieved at 0.08 mM in broken cell preparations of rat peritoneal mast cells. Tranilast also inhibited purified rat spleen and brain PGD synthetases. Furthermore, Tranilast prevented the PGD2 generation from intact mast cells stimulated by the calcium ionophore A23187. These results suggest that Tranilast exerts some of its therapeutic effects by prevention of PGD2 generation in mast cells and some other tissues.

Psoriasis and the arachidonic acid cascade.

Arachidonic acid (5.8,11,14-eicosatetraenoic acid C20:4, n-6) is released from the cell membrane by the action of phospholipases on membrane phospholipids. Metabolites of arachidonic acid, which are generically termed eicosanoids, including prostaglandins, thromboxane, leukotrienes and hydroxyeicosatetraenoic acids, have been implicated as mediators or modulators of a number of physiological functions and pathological conditions in both normal and diseased human skin. Particularly, eicosanoids have been suspected to play an important role in the pathogenesis of psoriasis, because a number of phenomena observed in psoriasis can be explained, at least in part, by the action of eicosanoids. This review will focus on recent progress regarding the significance of eicosanoids in the pathogenesis of psoriasis. Recent developments in the molecular biology in the eicosanoids have renewed interest in the role of eicosanoids in psoriasis. New understanding of the etiology of psoriasis and advances in its treatment due to recent progress in eicosanoid biology will also be presented.

Effect of botulinum C3 exoenzyme on cell growth and cytoskeleton organization in transformed human epidermal cells in culture: a possible role for rho protein in epidermal cells.

We examined the role of rho gene products (rho proteins) on cell growth and cytoskeleton organization in transformed human epidermal cells in culture (HSC-1), using recombinant botulinum C3 exoenzyme which specifically ADP-ribosylates rho proteins. Incubation of HSC-1 cell lysates with C3 exoenzyme revealed a single [32P]ADP-ribosylated protein with a molecular weight of 23,000. This protein was identified as rhoA protein by isoelectric focusing (pI 6.0). Addition of C3 exoenzyme to the culture medium of HSC-1 cells changed the shape of HSC-1 cells to a round form with beaded processes in a time- and dose-dependent manner. Moreover, C3 treatment reduced the cell growth rate; 72-h treatment with C3 exoenzyme at 1, 3, 10, 30 and 60 micrograms/ml culture medium resulted in 9.0 +/- 1.8%, 20 +/- 2.9%, 26 +/- 2.3%, 50 +/- 1.4% and 40 +/- 2.0% inhibition of the growth rate relative to controls, respectively. Under this condition, actin stress fibers were disassembled, as revealed using fluorescent-labeled phallacidin, whereas keratin intermediate filaments were not affected, visualized by immunofluorescence using anti-keratin antibody. These results suggest that rho proteins are closely related to cell growth and that these proteins regulate, at least in part, the assembly of actin stress fibers in transformed human epidermal cells.

Effect of cytotoxic prostaglandin, delta 12-prostaglandin J2 on E-cadherin expression in transformed epidermal cells in culture.

The cyclopentenone prostaglandins (PGs), such as delta 12-PGJ2 and PGA1, are potent inhibitors of growth in a variety of cultured cells, including human epidermal cells. To clarify the mechanism of the cytotoxicity of these PGs, we examined the effects of delta 12-PGJ2 on the function and expression of E-cadherin, which plays a major role in the maintenance of intercellular adhesion, in transformed human epidermal cells in culture (HSC-1). A 12-h incubation with 5 micrograms/ml of delta 12-PGJ2 did not affect the cell-binding activity of E-cadherin expressed in HSC-1 cells. Immunoblot analysis using a monoclonal antibody specific to human E-cadherin revealed that a 12-h incubation with 5 micrograms/ml of delta 12-PGJ2 induced E-cadherin expression in HSC-1 cells. Immunofluorescence using a monoclonal antibody against human E-cadherin demonstrated that E-cadherin was localized to the cell-cell contact regions in HSC-1 cells. Following a 12-h incubation with 5 micrograms/ml of delta 12-PGJ2, E-cadherin was also detected in a uniform pattern along cell junctions, although cell morphology was changed by the presence of cytotoxic PGs. These results suggest that the cytotoxicity of cyclopentenone PGs is related, at least in part, to E-cadherin expression in transformed human epidermal cells.

Induction of apoptosis, p53 and heme oxygenase-1 by cytotoxic prostaglandin delta12-PGJ2 in transformed endothelial cells.

Delta12-prostaglandin (PG)J2, which has been reported to have potent growth inhibitory activity in various tumor cells, induced apoptosis at 5 microg/ml culture medium in transformed mouse endothelial (F2) cells. Immunoblot analysis using anti-p53 or anti-WAF1 antibodies demonstrated that these two proteins had increased following delta12-PGJ2 treatment in F2 cells. Western blotting analysis using anti-heme oxygenase-1 (heat shock protein (HSP)32) antibody also revealed that delta12-PGJ2 induced HSP32 formation in F2 cells. HSP32 was also induced by heat shock treatment at 43 degrees C for 90 min. In contrast, HSP72 was not induced by heat shock or by delta12-PGJ2 treatment. In agreement with these findings, HSP32 immunofluorescence in the cytoplasm of F2 cells was intensified by delta12-PGJ2 treatment. More intense HSP32 immunoreactivity was similarly observed after heat shock treatment. These results suggest that delta12-PGJ2 caused the apoptotic cell death of F2 cells, which involved a certain process required for p53 or HSP32 induction.