Hypertrehalosemic hormone-regulated gene expression for cytochrome P4504C1 in the fat body of the cockroach Blaberus discoidalis.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 28:79-90 (1 995) Hypertrehalosemic Hormone-Regulated Gene Expression for Cytochrome P4504C1 in the Fat Body of the Cockroach, Blaberus discoidalis Kuang-Hui Lu, James Y. Bradfield, and Larry L. Keeley Laboratories for liiuertebrafe Neuroendocrirte Research, Departnlettt of Entomology, Texas A&M Utziurrsity, Texas Agriciilttiral Experiment Sfafioiz, College Station, Texas Hypertrehalosemic hormone (HTH) up-regulates expression of a gene for a cytochrome P450 of family 4 (CYf4CI) in the fat body of adult male B. discoi& / i s cockroaches. Studies were undertaken to determine the characteristics of HTH-dependent CYP4CI expression. A dot-blot assay was developed for routine measurement of the relative levels of CYP4C1-mRNA in fat body RNA extracts. A single injection of 10 pmol H T H produced a maximum CYf4CI response within 8 h. This dose corresponds with the dose needed for a maximum in vivo hypertrehalosemic response to HTH (Keeley et a[., 1991). Multiple treatments with HTH at 8 or 24 h intervals were no more effective than a single treatment for producing CYf4C7 expression. These results indicate that CYf4C1 expression i s sensitive to physiological doses of H T H and responds rapidly. CYP4C1 expression was suppressed by treatment with a-amanitin, an inhibitor of RNA polymerase II, but was unaffected by cycloheximide, an inhibitor of protein synthesis. HTH appears to influence CYP4C7 transcription without involvement of intervening regulatory genes. These results suggest that regulation of fat body CYf4CI expression is a major physiological action of HTH. @ 1995 Wiley-Liss, Inc. Key words: adipokinetic hormone, cockroach, cytochrome P450, fat body, gene expression Acknowledgments: This research was supported by National Science Foundation grant DCB9104536. Received December 3, 1993; accepted June 24, 1994. Address reprint requests to Larry College Station, TX 77843-2475. L. Keeley, Department of Entomology, Texas A&M University, Kuang-Hui Lu is now at Department of Entomology, National Chung-Hsing University, Taichung, Taiwan, ROC. 0 1995 Wiley-Liss, Inc. 80 Lu et al. INTRODUCTION Cytochrome P450 enzymes are a superfamily of monooxygenases for which more than 200 CYP genes are described (Nelson et al., 1993). P450 enzymes have two main functions in animals. First, P450 enzymes degrade toxic chemicals such as drugs, insecticides, environmental pollutants, and natural plant products. Second, they metabolize natural physiological substrates such as steroids, fatty acids, and arachidonic acid (Juchau, 1990). The expression of many CYP genes is induced by xenobiotics or hormones (Gonzalez, 1989; Lund et al., 1991). The hypertrehalosemic hormone (HTH) was isolated and sequenced from the neotropical cockroach Blaberus discoidalis (Hayes et al., 1986) and belongs to the family of insect and crustacean adipokinetic hormone/red pigment-concentrating hormones (AKH/RPCH) (Gade, 1990).HTH regulates fat body metabolism in B. discoidalis to increase glycogenolysis for trehalose biosynthesis and for elevation of hemolymph carbohydrate (Lee and Keeley, 1994a). In addition, HTH stimulates fat body heme synthesis and enhances the rate of juvenile hormone-dependent protein synthesis in the fat body of vitellogenic females (Keeley et al., 1991). In studies on the cellular action of HTH, it was observed that HTH increased [3Hluridineincorporation into fat body RNA and a-amanitin suppressed HTH-related heme synthesis (Lee and Keeley, 199413).Furthermore, in vitro translation of fat body RNA from HTHtreated animals revealed increased synthesis of three polypeptides when compared to RNA from Ringer-treated controls. These results suggested that HTH stimulated specific gene expression in addition to its direct actions on trehalose synthesis. Differential screening of a fat body cDNA library prepared from HTHtreated cockroaches identified an HTH-sensitive gene that was cloned, sequenced, and characterized as a cytochrome P450 gene (Bradfield et al., 1991). This P450 gene was identified as a member in family 4, as the first representative of a new subfamily C, and was designated CYP4CI (Nebert et al., 1991). CYP4CI regulation by HTH represents the first example of a specific neurohormonal regulation of gene expression in insects and provides an excellent model in which to study the mode of action of AKHHTH neurohormones on fat body metabolism. HTH exerts an almost immediate stimulation of fat body trehalose biosynthesis through activation of enzymes related to glycogenolysis, but at the same time produces a longer term stimulation of CYP4CZ expression. Are both actions part of the same second messenger cascade or are two separate signal transduction mechanisms involved-one for immediate enzyme activation and a second for gene regulation? In this study, we have characterized the regulation of CYP4C1 expression by HTH. CYP4C1-mRNA levels were maximally elevated within 8 h after exposure to a single physiological dose of HTH and were sensitive to the inhibition of RNA polymerase I1 but not to the inhibition of protein biosynthesis. These results suggest that stimulation of CYP4CZ expression is a significant regulatory action by HTH for fat body metabolism in B. discoidalis. HTH-Regulated P450 Gene Expression 81 MATERIALS AND METHODS Experimental Animals B. discoidalis were reared in wood shavings in 40 liter plastic buckets, fed dry dog food and water ad libitum, and kept in a 12-h 1ight:lZh dark circadian cycle at 27 & 2°C (Keeley, 1971). Newly emerged (day 0) adults were collected daily, separated according to gender, and housed in plastic rat cages under rearing conditions until used for experiments. The day before being used in experiments, animals were decapitated to eliminate the corpora cardiaca, the source for endogenous HTH, and the wound was sealed with hot beeswax:petroleum jelly (1:l). Injection Regimen Experiment animals received HTH in 10 pl of insect Ringer (Ephrussi and Beadle, 1936) by injection with a microsyringe through a ventral abdominal intersegmental fold. Controls received 10 p1 Ringer in the same manner. Dot-Blot Analysis Fat bodies were pooled from 3-4 experimental animals, and total RNA was extracted (Puissant and Houdebine, 1990). One microgram of total RNA from each treatment sample was denatured with formaldehyde (White and Bancroft, 1982) and loaded into the wells of a dot-blot Minifold@ I (Schleicher & Schuell, Keene, NH) containing a positively charged ZetaProbe nylon membrane (BioRad, Richmond, CA). The blotted membranes were rinsed with l x SET (20x = 3.0 M NaC1; 20 mM EDTA; 0.5 M Tris-C1, pH 7.5), and RNA was cross-linked to the filter by illumination with ultraviolet (304 nm) for 5 min. For a hybridization probe, cloned CYP4CZcDNA was labeled using [ C X - ~ ~ P ] ~and C T Pa random priming method (DECAprimeTMDNA labeling kit; Ambion Inc., Austin, TX). Hybridization was in 4x SSPE (20x = 3.0 M NaC1; 0.2 M Na-phosphate buffer, pH 7.4; 20 mM EDTA) and 2% SDS at 60°C overnight; washes were with 0 . 1 ~ SSPE, 1%SDS at the same temperature. Each hybridized dot was excised and its radioactivity quantitated by liquid scintillation spectrometry. Therefore, all values reflect the proportional amount of CYP4Cl-mRNA per microgram of total fat body RNA. The specificity of our CYP4C1-cDNA probe was demonstrated previously by Northern blotting of fat body total RNA (Bradfield et al., 1991). A titration experiment was performed wherein samples of fat body total RNA (0-1.0 pg in increments of 0.1 pg) were applied to nylon membrane and hybridized with CYP4CZ-cDNA as described above. To compensate for possible quenching, heterologous RNA (poly(A)RNA from adult female Manduca sextn fat body) was added such that each sample contained 1.0 pg total RNA. The relationship of bound probe (cpm) to CYP4C1-mRNA was linear (r2 = 0.98) throughout the range. Therefore, expression of the data in cpm provides a close estimate of the relative abundance of the CYP4C1 message within each experiment. However, because specific activity of the probe fluctuated slightly during the course of these studies, cpm values are not directly comparable between figures unless otherwise noted. 82 Lu etal. Inhibitors of mRNA and Protein Biosynthesis CYP4C1-mRNA was measured after treatment with a-amanitin to inhibit fat body mRNA synthesis. Amanitin and 100 pmol HTH were administered sequentially to test animals in 10 p1 of Ringer. Control insects were injected twice with 10 p1 Ringer. Fat bodies were isolated after 8 h, and RNA was extracted for analysis. CYP4C1-mRNA was measured after inhibition of fat body protein synthesis with cycloheximide (CHXM). Experimental animals were treated with 100 pg CHXM to suppress fat body protein synthesis, and 100 pmole HTH was administered 1 h later. A second 100 yg dose of CHXM was administered 4 h after HTH to insure continued suppression of fat body protein synthesis. Fat bodies were isolated 8 h after the HTH treatment, RNA was isolated, and CYP4C1 transcript levels were determined. The inhibitory action of the CHXM treatments was confirmed by administering 10 yCi ["S]methionine in place of HTH to CHXM-treated insects and determining the extent of 35S-labelincorporated into fat body proteins during the 8 h study period. Fat body proteins were precipitated, lipid extracted, and redissolved in 1 N NaOH for liquid scintillation spectrometry and protein determination (Keeley et al., 1988). Fat body protein synthesis was suppressed by 87% in animals treated with CHXM compared to Ringer-treated controls. No animals died during the 8 h study period after treatment with any of the doses of a-amanitin or CHXM. Animals treated with a-amanitin remained normally active; animals treated with CHXM became sluggish. Chemicals All chemicals were reagent grade. CHXM and a-amanitin were purchased from Sigma Chemical Co. (St. Louis, MO); [a-32PldCTPwas obtained from NEN-DuPont (Boston, MA). HTH was synthesized and standardized by the Texas A&M University System, Biotechnology Support Laboratory. Statistics Means or experimental and control groups were based on three independent samples. Each sample consisted of the RNA extracted from 3 4 pooled fat bodies from individual animals. Two aliquots from each RNA extract were blotted and probed and the cpm averaged for a single sample value. Means for experiments with multiple variables were analyzed by ANOVA followed by a Tukey-Kramer multiple comparisons post-test to show statistical differences between individual means. An unpaired Students t-test was used when statistical analysis involved comparing means between only single experimental and control groups. Statistical analyses were performed using InStat 2.0@(GraphPad Software, San Diego, CA) for Apple Macintosh computers. RESULTS Expression Profiles of CYP4CZ in Response to HTH CYP4C1 transcript was measured in fat body of decapitated adult males at intervals during 24 h following injection of 100 pmol HTH (Fig. 1).CYP4C1- HTH-Regulated P450 Gene Expression -0- 83 Control +HTH .. . . \ 4P *0 .... . - - -.- - ' I * * * . I * * * * - Fig. 1 , CYP4C1 transcript accumulation in response to HTH in fat body of decapitated, 1 day-old adult male B . discoidalis. Values represent mean f SEM for three samples; each sample consisted of pooled fat bodies from four animals. Animals were decapitated on day 0 and injected on day 1 with 100 pmol HTH in 10 1.11 Ringer. Fat bodies were removed for RNA extraction at the designated time intervals after HTH administration. Control insects were injected with 10 pI Ringer. mRNA levels did not change significantly in Ringer-treated control animals during the 24 h observation period. By comparison, CYP4C1-mRNA increased rapidly in HTH-treated animals and was twice that of control animals by 4 h. A maximum 2.75-fold increase in transcript levels was attained at 8 h. This peak level persisted through 12 h and then began to decline, and according to linear extrapolations of the rate of decline, CYP4C1-mRNA would have returned to basal levels by 40 h. Effect of Multiple Treatments with HTH on CYP4C1 Expression Previous studies used three daily injections of HTH to produce maximum CYP4C1 expression (Bradfield et al., 1991). The present experiment was undertaken to determine if repeated HTH treatments resulted in greater accumulation of CYP4Cl-mRNA. Decapitated animals were injected daily with 100 pmol HTH for 1, 2, or 3 days. The results show that HTH stimulated CYP4C1 transcript accumulation by two- to three-fold over Ringer-treated control animals for all injection regimens (Fig. 2A). No significant differences were observed in the levels of CYP4C1-mRNA at 24 h after the last HTH treatment, regardless of the number of treatments. The results shown in Figure 1 indicate that maximal CYP4C1 transcript levels were attained 8 h after HTH administration. Therefore, injections of 100 pmol HTH were administered at 8 h intervals to determine if additional exposures to hormone at the time of maximum response reinforced CYP4Cl 84 Lu et al. One Treatment Two Treatments Three Treatments 0 100 0 100 200 300 400 CYP4C 1-mRNA (cprn) 500 600 300 500 10 One Treatment Two Treatments Three Treatments 200 400 CYP4C1-mRNA (cpm) Fig. 2. Effects of multiple HTH treatments on CYP4Cl expression in decapitated, 1-day-old adult male 6 discoidalis. Values represent mean f S E M for three samples; each sample consisted of pooled fat bodies from four animals. All animals were decapitated on day 0. A: Animals were injected with 100 pmol HTH in 10 PI Ringer on days 1, 2, andlor 3 with measurements of CYP4C1 -mRNA 24 h after the last injection. 6: Animals were injected with 100 pmol HTH in 10 pI Ringer at 8, 16, and/or 24 h with measurements of CYP4C1-mRNA 8 h after the last injection. Control animals received 10 pl Ringer at the same time intervals. Both the daily and the 8 h membranes were hybridized together so that cpms presented in both A and B are comparable, transcript accumulation (Fig. 28). Although values for CYP4Cl-mRNA did increase modestly with increasing injections, the increases were not statistically significant. Therefore, no additional accumulation of transcript was evident in response to multiple 8 h treatments with HTH as compared to a single treatment. HTH-Regulated P450 Gene Expression 85 Effects of Transcriptional/TranslationalInhibitors on CYP4C1 Expression Experiment animals were treated with a-amanitin to determine if inhibition of DNA-dependent RNA polymerase-I1 suppressed HTH-dependent CYP4CZ expression. Animals were injected with four different doses of aamanitin along with 100 pmol HTH; CYP4C1-mRNA levels were measured in the fat body 8 h later. HTH (100 pmol) increased CYP4C1-mRNA by twofold over control animals. All doses of a-amanitin at or above 1.25 pg suppressed CYP4C1-mRNA to control levels in HTH-treated animals (Fig. 3A). CHXM was used to determine if protein synthesis was required for HTHdependent CYP4C1 expression. The CHXM treatment did not suppress CYP4CZ expression in HTH-treated, decapitated animals (Fig. 3B); instead, CHXM appeared to increase CYP4Cl-mRNA levels compared to Ringertreated control animals. Dose Response of CYP4C1 Expression to HTH Treatments HTH dose sensitivity was measured for CYP4CZ expression. Test doses of HTH were administered to day 1, decapitated adult male B. discoidalis, and the amount of fat body CYP4Cl-mRNA was determined 8 h later. CYP4C1mRNA increased in a dose-dependent manner between 0.65 and 11 pmol HTH per animal (Fig. 4). The ED5(,dose was 3 pmol HTH. DISCUSSION The relationship between HTH and CYP4C1 expression in B . discoidalis constitutes the first insect examples for both neurohormone-regulated gene expression and endocrine-sensitive cytochrome P450 genes. CYP4CZ was discovered based on the abundance of its transcript in the fat body of HTHtreated cockroaches relative to HTH-deprived animals (Bradfield et al., 1991). However, the early experiments employed high doses of HTH administered daily for several days and did not examine the sensitivity of CYP4C1 expression to HTH or the kinetics of transcript accumulation. The present research defines more precisely the temporal relationship between HTH action and CYP4C1 expression. CYP4CZ expression occurs rapidly in response to HTH. A measurable increase in transcript levels was observed within 2 h after treatment with HTH, and a maximum level was attained by 8 h (Fig. 1).There are no other reports of neuropeptide-responsive gene expression in insects to which these times for response can be compared, by 2 h appears to represent a rapid hormonedependent response. For example, juvenile hormone (JH) induces vitellogenin gene expression in Locusta rnigvatovia fat body, and vitellogenin mRNA is not detected until 18 h after JH treatment (Chinzei et al., 1982; Wyatt, 1988). The rapidity of the CYP4CZ response suggests that HTH likely regulates the CYP4CZ gene in a relatively direct manner. Multiple doses of HTH did not cause accumulation of CYP4C1-mRNA above the optimal level detected at 8 h after a single treatment (Fig. 2). In our initial discovery of CYP4CZ expression, we used a 3 day regimen of HTH injections (Bradfield et al., 1991). It was hypothesized that multiple daily in- 86 Lu et al. Control 100 pmole HTH HTH + 0.63 pg Amanitin HTH + 1.25 pg Amanitin HTH + 2.50 pg Amanitin HTH + 5.00 c(gAmanitin I 0 400 200 600 . . . I . 800 CYP4Cl-mRNA (cpm) (6) 0 50 100 150 200 250 CYP4C1-mRNA (cpm) Fig. 3 . Effects of a-amanitin (A) and cycloheximide (CHXM) (B) on HTH-related CYP4CI expression in the fat body of decapitated, 1 -day-old adult male R. discoidalis. Values represent mean 2 SEM for three samples; each sample consisted ot pooled fat bodies from four animals. jections might produce transcript accumulation exceeding that obtained with a single dose; however, this was not the case, as transcript levels remained essentially constant despite repeated administration of HTH. The data in Figure 2 were obtained using the same radiolabeled probe preparation so that the cpm can be compared between the 24 h (Fig. 2A) and 8 h (Fig. 2B) injection regimens. The results appear to suggest that the amount of mRNA present at 24 h was the same as the amount present at 8 h which was maximal. This is a confounding result since by 24 h the transcript level should have declined by 50% based on the results shown in Figure 1. We have no explanation for this apparent discrepancy. It is possible that the values for one and two 24 h treatments are in fact abnormally high, by chance, and should have been closer to the value of the three 24 h treatments which was approxi- HTH-Regulated P450 Gene Expression -1300 L 87 I - -- 100 I I I I I I I I I N15 0 10' 100 10' 10' 102 1 pmol HTH Fig. 4. HTH dose response for CYP4CI expression in the fat body of decapitated, 1-day-old adult male 5. discoidalis. Values represent mean SEM for five samples; each sample consisted of pooled fat bodies from three animals. Decapitated male 6. discoid;tlis were injected with the indicated dose of HTH in 10 pI Ringer; fat bodies were removed after 8 h and pooled, and the RNA was extracted and CYP4C1 -mRNA determined. Dose responses indicated by the dotted lines are as follows: minimum = 0.65 pmol HTH; maximum = 11 pmol HTH; EDSO= 3 pmol HTH. * mately 50% lower and closer to the expected value. Finally, fat body CYP4C1mRNA levels were increased two to three-fold over controls by 8 h after a single HTH treatment, but these elevated transcript levels were not augmented by repeated treatments with HTH at 8 h intervals (Fig. 2B). This is also a confounding result since 8 h is maximal for transcript accumulation, and we would anticipate that continued stimulation of CYP4CI expression by repeated HTH injections at 8 h intervals would produce ongoing transcript accumulation. Both of these results suggest that there is an upper limit for HTH stimulation of pretranslational CYP4C1 gene expression beyond the constitutive levels. Since we have little information on how HTH induces CYP4C1, it is possible that a second messenger cascade is involved that may include feedback controls. Inhibitors of RNA polymerase I1 and protein biosynthesis were used to examine the mechanisms of CYP4C1 gene regulation by HTH. a-amanitin completely blocked the HTH-dependent increase in fat body CYP4Cl-mRNA (Fig. 3A), indicating that the increase in fat body CYP4C1-mRNA was due to HTH stimulation of CYP4C1 transcription. On the other hand, the CHXM treatment regimen strongly inhibited fat body protein synthesis but did not inhibit the HTH-dependent increase in CYP4C1-mRNA (Fig. 3B). The latter results suggest that HTH promoted CYP4CZ expression independent of the translation of any regulatory products from other genes. Blocking protein synthesis with CHXM surprisingly increased CYP4ClmRNA significantly compared to Ringer-treated controls. A similar increase 88 Lu et a]. in female-specific P4503A1-mRNA was observed in the liver of rats after CHXM treatment (Burger et al., 1990). The increase in CYP4C1-mRNA after CHXM treatment suggests that noninduced fat body might synthesize proteins that suppress CYP4CZ expression or degrade CYP4C1-mRNA. If CHXM inhibited the synthesis of such putative proteins, CYP4Cl transcription might increase and/or transcripts might accumulate due to reduced degradation. The dose-response relationship was determined for HTH and CYP4C1 expression. In a previous study, 100 pmols HTH were used to insure a maximum response (Bradfield et al., 1991). This was based on earlier in vivo dose-response stimulation of fat body heme biosynthesis between 10 and 100 pmols HTH per animal (Keeley et al., 1991).The present studies showed that 0.65-11 pmols HTH was the dose-response range for CYP4CZ expression (Fig. 4). In vivo trehalose biosynthesis is dose-dependent between 1 and 10 pmoles of HTH per animal for adult male B. discoidulis (Keeley et al., 1991). Therefore, within the limits of experimental variability, the HTH dose response for CYP4CZ expression likely coincides with the physiological range for HTHdependent trehalose biosynthesis. In general, we observed that HTH stimulates both trehalose and heme biosynthesis by two- to four-fold in B. discoidalis fat body (Keeley et al., 1991; Lee and Keeley, 1994a). This agrees with the magnitude of the CYP4CZ response to HTH. The rapidity, sensitivity, and magnitude of the CYP4CI response relative to other HTH-sensitive physiological events suggest that HTH stimulation of CYP4C1 expression is a direct action by the hormone and not a delayed, secondary effect that occurs subsequent to other more primary actions by the hormone. Therefore, CYP4C1 expression is likely a significant physiological effect by HTH for the production of P4504C1, which is somehow related to the hypertrehalosemic action of the hormone. As a peptide, HTH probably does not enter the cell and interact directly with the CYP4CZ gene; rather the gene expression is probably implemented through a presently undefined HTH-dependent second messenger transduction cascade. The nature of this cascade remains undetermined. Past studies on HTH second messengers have demonstrated that HTH does not increase fat body CAMPnor affect adenylyl cyclase activity (Lee and Keeley, 1994a). Similarly, cGMP analogs have no effects on trehalose biosynthesis, but both extra- and intracellular Ca2' are important for maximal HTH-dependent hypertrehalosemic activity (unpublished results). It is of interest to determine if HTH stimulation of CYP4CZ expression is an extension of the second messenger cascade that stimulates trehalose biosynthesis or is a separate transduction cascade. The enzymology of the P4504C1 protein is under investigation in our laboratory but is not yet elucidated. Based on mammalian research, CYP4 enzymes perform o-oxidation of medium-chain fatty acids such as lauric and arachidonic acids (Bains et al., 1985; Hardwick et al., 1987). HTH is a member of the AKH/RPCH family, and a major action of AKHs is to mobilize fat body fatty acids (Mayer and Candy, 1967) and to enhance fatty acid metabolism (Robinson and Goldsworthy, 1974, 1977). 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