Functional alteration of guanine nucleotide binding proteins (Gs and Gi) in psoriatic epidermis

Journal of Dermatological Science 17 (1998) 61 – 66

Functional alteration of guanine nucleotide binding proteins (Gs and Gi) in psoriatic epidermis T. Tamura, H. Takahashi, A. Ishida-Yamamoto, Y. Hashimoto, H. Iizuka * Department of Dermatology, Asahikawa Medical College, 3 -11 Nishikagura, Asahikawa 078, Japan Received 26 May 1997; received in revised form 29 August 1997; accepted 16 September 1997

Abstract Psoriatic involved epidermis has been characterized by a defective b-adrenergic adenylate cyclase response. It is also characterized by increased cholera toxin- and forskolin-induced cyclic AMP accumulations. Due to the fact that receptor signals are transduced to adenylate cyclase through guanine nucleotides binding proteins (G-proteins), that affect cholera toxin- and forskolin-induced cyclic AMP accumulations, possible alterations of G-proteins of psoriatic involved and perilesional uninvolved epidermis by using toxin-catalyzed ADP-ribosylation and immunoblot analyses was investigated. Cholera toxin catalyzes ADP-ribosylation of stimulatory guanine nucleotides binding protein (Gs) in either trimeric (inactive) or monomeric (active) form, while islet activating protein (IAP) catalyzes ADP ribosylation of inhibitory guanine nucleotide binding protein (Gi) in only trimeric (inactive) form. Results indicate that although the psoriatic involved epidermis shows increased cholera toxin- and IAP-catalyzed ADP-ribosylations, the amounts of immunoreactive Gsa or Gia are not significantly altered. The increase in IAP-catalyzed ADP-ribosylation indicates increased inactive Gi, explaining the increased forskolin-induced cyclic AMP accumulation. The increase in cholera toxin-catalyzed ADP-ribosylation of Gs explains the increased cholera toxin-induced cyclic AMP accumulation in the psoriatic involved epidermis. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Adenylate cyclase; Psoriasis; G-protein; ADP-ribosylation; Cholera toxin; Islet activating protein

1. Introduction Adenylate cyclase is a membrane-bound enzyme that catalyzes the conversion of ATP to cyclic AMP. The adenylate cyclase complex is * Corresponding author. Tel: + 81 166 652111; fax: + 81 166 657751.

composed of three functionally-distinct, membrane-bound components: (1) the receptor component; (2) the guanine nucleotide binding proteins (G-proteins); and (3) the catalytic subunit. The stimulation or inhibition of receptoradenylate cyclase systems is mediated by stimulatory and inhibitory G proteins (Gs and Gi), respectively [1–4]. These G-proteins are het-

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erotrimeric and composed of a, b and g subunits. The a subunits of Gs and Gi contain sites for nicotinamide adenine dinucleotide (NAD)-dependent ADP-ribosylation catalyzed by bacterial toxins. Cholera toxin catalyzes ADP-ribosylation of Gsa, resulting in sustained Gsa stimulation. Isletactivating protein (IAP), one of the pertussis toxins, catalyzes ADP-ribosylation of Gia, resulting in the inhibition of the Gia function. It has been reported that epidermis contains both Gsa and Gia (Gi2a and Gi3a) that is modified by these bacterial toxins [3,4]. Psoriatic involved epidermis has been characterized by a defective b-adrenergic adenylate cyclase response [5,6]. It is also characterized by increased cholera toxin- and forskolin-induced cAMP accumulations [7]. Although the defective b-adrenergic response has been attributed to a decreased b-adrenergic receptor and/or functional uncoupling of the receptor and Gs [8 – 11], the detailed mechanism of the increased cholera toxin-, and forskolin-induced cyclic AMP accumulations remains unknown. In the present study, in order to determine whether these alterations of cyclic AMP responses are associated with derangements of G-proteins in the psoriatic involved epidermis, toxin-catalyzed ADP-ribosylation and immunoblot analyses has been performed.

2. Materials and methods

2.1. Patients All patients are male, their ages ranged from 36 to 68 years (mean 49 years old). Duration of the disease ranged from 1 to 10 years (mean 5.5 years), psoriasis area and severity index (PASI) score ranged from 13.5 to 15.8 (mean 14.3).

2.2. Preparation of epidermis Epidermal samples were obtained from backs of four adult male patients. No active treatment was given for at least 1 week before taking the skin. After 0.5% lidocaine anesthesia, sheets of skin were obtained by keratome biopsy set at 0.3

mm. The epidermal sheets, that contain small amounts of melanocytes and Langerhans cells, were homogenized in 20 mM Tris–HCl (pH 7.5) containing 1 mM ethylenediaminetetraacetic acid (EDTA). The homogenate was centrifuged at 10000× g for 10 min, and the resulting precipitate was resuspended in the buffer. These washing procedures were repeated twice at 4°C. The membrane preparation was then suspended in the above-mentioned buffer at a final concentration of 10 mg protein/ml. Solubilization of epidermal cell membranes was accomplished by addition of sodium cholate and NaCl at final concentrations of 1% (w/v) and 100 mM, respectively. After vigorously vortexing and incubating for 1 h on ice, the supernatant obtained by centrifugation at 25000× g for 30 min was used for further experiments as cholate extracts.

2.3. Cholera toxin-catalyzed and IAP-catalyzed ADP-ribosylation [3,4] Cholate extracts (500 mg of protein) were incubated for 1 h at 30°C with 10 mM [a-32P]NAD (1000 Ci/mmol) in the presence of 50 mM phosphate buffer (pH 7.6), 10 mM thymidine, 1 mM ATP, 3 mM phosphoenolpyruvate, 1 mM EDTA, 0.1 mM GTP, 1 mM MgCl2, 1 mM dithiothreitol (DTT) and 75 mg/ml preactivated cholera toxin with a total volume of 100 ml. Cholera toxin was preactivated by incubation with 30 mM DTT and 1 mM adenosine triphosphate (ATP) for 15 min at 30°C. The cholate extracts were incubated with 148 kBq or 4 mCi [a-32P]NAD (37 TBq/mmol) in the presence of 50 mM phosphate buffer (pH 7.6), 1 mM EDTA, 1 mM ATP, 1 mM DTT, 10 mM thymidine, 3 mM phosphoenolpyruvate, 0.1 mM guanosine triphosphate (GTP), 2.5 mM MgCl2, and 75 mg/ml activated cholera toxin with a total volume of 100 ml for 3 h at 30°C. IAP was preactivated by incubation with 50 mM ATP, 10 mM DTT, and 50 mM Tris–HCl (pH 7.5) at 30°C for 15 min. The cholate extracts were incubated for 3 h at 30°C with 4 mCi [a-32P]NAD in the presence of 100 mM Tris–HCl (pH 8.0), 1 mM EDTA, 1 mM ATP, 1 mM DTT, 10 mM thymidine, 3 mM phosphoenolpyruvate, and 20

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mg/ml IAP with a total volume of 100 ml. The ADP-ribosylation reaction was terminated by the addition of an equal volume of Laemmli sample buffer (1% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 10% glycerol, 62.5 mM Tris – HCl, and 0.02% bromophenol blue, pH 6.8) [12], and by heating for 2 min at 100°C.

2.4. Polyacrylamide gel electrophoresis and autoradiography Cholate extracts (40 mg of protein each) were loaded on each lane of a 1-mm-thick polyacrylamide slab gel, consisting of a 12.5% separation gel and 4.5% stacking gel, and subjected to electrophoresis as described by Laemmli [12]. After electrophoresis, the gels were stained with Coomassie brilliant blue R-250, destained, and dried on Whatman 3 MM filter paper. The dried gels were autoradiographed at −80°C for 24 h using Kodak X-Omat film with an intensifying screen. Molecular weight markers were: phosphorylase b (94000), bovine serum albumin (67000), ovalbumin (43000), carbonic anhydrase (30000), trypsin inhibitor (20100), and a-lactalbumin (14400). The gels were dried and the density was analyzed with a BAS 2000 autoradio image analyzer (Fuji fix, Tokyo, Japan). The experiments were performed in duplicates, each time essentially with the same results.

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2.6. Statistical analysis Student’s t-test (paired t-analysis) was used for statistical analysis of data.

2.7. Materials Anti-G protein polyclonal antibodies (RM/1 and AS/7) were purchased from DuPont (Wilmington). RM/1 is specific to Gsa [13], while AS/7 is specific to Gia1,2 [14]. AS/7 also recognizes transducin [14] that does not present in keratinocytes. Cholera toxin and phosphoenol pyruvate were obtained from Sigma (St Louis, MO). IAP was purchased from Funakoshi Chemicals (Tokyo, Japan). All other chemicals were obtained from Nakarai Tesque (Kyoto, Japan).

3. Results and discussion Investigated were G-proteins of the psoriatic involved and perilesional uninvolved epidermis by toxin-catalyzed ADP-ribosylation and by immunoblot analyses. Cholera toxin catalyzed ADPribosylation of 45 kDa membrane protein and IAP catalyzed ADP-ribosylation of 40 kDa membrane protein, respectively (Figs. 1 and 2). Although the same amount of protein (40 mg) was loaded for comparison, both cholera toxin- and

2.5. Immunoblot analysis Following the SDS-PAGE (40 mg of protein each), gel slabs containing separated proteins were electrophoretically transferred to nitrocellulose membrane in Tris-buffered saline (TBS) containing 25 mM Tris–HCl and 192 mM glycine. Blotting detection kit-for human antibody (Amersham, Bucks., UK) was used for immunodetection. Commercially available Gs and Gi antibodies were diluted 1000-fold by saline. The density of the blot was analyzed by a BAS 2000 autoradioimage analyzer. The experiments were performed in duplicates, each time essentially with the same results.

Fig. 1. Cholera toxin-catalyzed ADP-ribosylation of psoriatic involved and uninvolved epidermis. Membrane preparation (cholate extracts) were incubated with [a-32P]NAD in the presence of 75 ng/ml cholera toxin for 3 h. A total of 40 mg of protein was applied in each lane of SDS-PAGE (I, involved epidermis; U, uninvolved epidermis).

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Fig. 2. IAP-catalyzed ADP-ribosylation of psoriatic involved and uninvolved epidermis. Membrane preparation (cholate extracts) were incubated with [a-32P]NAD in the presence of 20 ng/ml IAP for 3 h. A total of 40 mg of protein was applied in each lane of SDS-PAGE (I, involved epidermis; U, uninvolved epidermis).

IAP-catalyzed ADP-ribosylations of the psoriatic involved epidermis were significantly increased compared to the uninvolved epidermis (P B 0.01 and PB0.05, respectively) (Figs. 1 and 2). Neither immunoreactive Gsa (45 kDa and 52 kDa proteins) nor Gia (40 kDa protein), however, were significantly altered in the psoriatic involved epidermis (Figs. 3 and 4). Although only 45 kDa membrane protein was

Fig. 3. Immunoblot of membrane proteins from involved or uninvolved psoriatic epidermis. Samples (40 mg of protein, respectively) were separated on a SDS-PAGE before electrophoreic transfer to nitrocellulose membrane. The sheets were incubated with polyclonal anti-Gsa antibodies (I, involved epidermis; U, uninvolved epidermis).

Fig. 4. Immunoblot of membrane proteins from involved or uninvolved psoriatic epidermis. Samples (40 mg of protein, respectively) were separated on a SDS-PAGE before electrophoreic transfer to nitrocellulose membrane. The sheets were incubated with polyclonal anti-Gi2a antibody that detects both Gi2a and Gi3a present in keratinocytes [17] (I, involved epidermis; U, uninvolved epidermis).

detected by cholera toxin-catalyzed ADP-ribosylation (Fig. 1), 52 kDa membrane protein was also detected by immunoblot for Gsa (Fig. 3). Most tissues including cultured fetal rat skin keratinocytes (FRSK cells) are known to contain two major bands of 45 kDa and 52 kDa, both of which are ADP-ribosylated by cholera toxin [15]. In pig and human epidermis, however, only 45 kDa Gsa has been detected by ADP-ribosylation analysis [4]. Due to the fact that ADP-ribosylation is associated with the stimulation of Gs, the 45 kDa Gsa seems to be the functional Gs in the psoriatic epidermis; 45 kDa Gsa has been known to be more functionally-efficient than the 52 kDa Gsa in other tissues [16]. Consistent with that epidermal keratinocytes express only two types of Gia2,3 [17] and that the antibody, AS/7, is specific to Gia1,2 [14], only a single 40 kDa band, that corresponds to Gia2, was detected by the immunodetection (Fig. 4). Although only a single ADP-ribosylation band was appreciated by IAP (Fig. 2), it is possible that Gia3 is actually increased in the psoriatic epidermis. Without the specific antibody to Gia3, the answer to this question remains to be determined. It has been known that IAP catalyzes ADP-ribo-

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sylation when Gi is only trimeric (inactive) form [4]. The increased IAP-catalyzed ADP-ribosylation (without an alteration of immunoreactive Gia), therefore, suggests that Gi is mostly inactive in the psoriatic involved epidermis; this explains the increased forskolin-induced activation of adenylate cyclase that is under the depressive tonus of Gi physiologically. On the other hand, cholera toxin catalyzes ADP-ribosylation of both trimeric (inactive) and monomeric (active) forms of Gsa [3]. Due to the fact that immunoreactive Gsa (both trimeric and monomeric forms) was not significantly altered in the psoriatic epidermis, the mechanism of the increased cholera toxin-catalyzed ADP-ribosylation requires further clarification. It has been known that the cholera toxin-catalyzed ADP-ribosylation depends on another cytosolic factor, ARF (ADP-ribosylation factor), that has no effect on IAP-catalyzed ADP-ribosylation [18]. Although it is tempting to speculate that ARF level is altered (increased) in the psoriatic involved epidermis, resulting in the increased cholera toxincatalyzed ADP-ribosylation as well as an increased cholera toxin-induced cyclic AMP accumulation [7], no data are available regarding the amounts of ARF in the psoriatic epidermal tissues. It has long been known that the psoriatic involved epidermis shows a defective b-adrenergic adenylate cyclase response, that is accompanied by increased cholera toxin- and forskolin-induced cAMP accumulations. The results indicate an increased functional (cholera toxin-stimulated) Gs activity and an increased inactive (trimeric) form of Gi in the psoriatic involved epidermis. The former explains the increased cholera toxin-induced, and the latter explains the increased forskolin-induced cAMP accumulations. Besides the psoriatic involved epidermis, hyperproliferative epidermis has been known to show similar adenylate cyclase responses, that include the defective b-adrenergic adenylate cyclase response [19,20]. It is interesting to note that tape-stripping induced and tumor promoter-treated hyperproliferative epidermis shows similar alterations of Gprotein functions such as increased cholera

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toxin-catalyzed ADP-ribosylation of Gsa, increased cholera toxin- and forskolin-induced cAMP accumulations [3,4,9].

Acknowledgements This study was supported in part by Grants 08457233 (H.I.), 08770617 (T.T.), 08770618 (H.T.), 08670940 (A. I-Y), and (Y.H.) from the Ministry of Education, Science, Sports, and Culture of Japan and from the Ministry of Health and Welfare, Japan.

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