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Estrogen Increases CD38 Gene Expression and Leads to Differential Regulation of Adenosine Diphosphate (ADP)-Ribosyl Cyclase and Cyclic ADP-Ribose Hydrolase Activities in Rat Myometrium¹

^a Departments of Veterinary PathoBiology, ^c Pediatrics, ^b Pharmacology, Colleges of Veterinary Medicine and Medicine, University of Minnesota, St. Paul, Minnesota 55108

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Hormones influence uterine contractility through their effects on intracellular calcium. The regulation of intracellular calcium in uterine smooth muscle is achieved by several mechanisms and includes mobilization from intracellular stores by inositol 1,4,5-trisphosphate and ryanodine-sensitive channels. Cyclic ADP-ribose (cADPR), a metabolite of NAD⁺, is known to mediate calcium release through ryanodine receptor channels. A cell surface glycoprotein, CD38, catalyzes the synthesis and breakdown of cADPR and thus possesses bifunctional enzymatic activity. The regulation of cADPR synthesis by ADP-ribosyl cyclase (cyclase) or degradation by cADP-ribose hydrolase (hydrolase) by hormones in the myometrium is poorly understood. We investigated the effects of estradiol-17ß on CD38 expression and the synthesis and degradation of cADPR in myometrial smooth muscle obtained from ovariectomized rats. CD38 expression was studied by reverse transcription polymerase chain reaction and Western blot analyses. In uterine microsomal fractions, cyclase and hydrolase activities were measured using nicotinamide guanine dinucleotide and [³²P]cADPR as substrates, respectively. Microsomal proteins subfractionated by SDS-PAGE and gel filtration were used to determine the fractions containing cyclase and hydrolase activities. The results demonstrate that cyclase and hydrolase activities are associated with a single protein fraction, similar to CD38 in uteri from both ovariectomized and estradiol-treated rats, and estradiol-17ß causes 1) increased CD38 mRNA and protein expression and 2) significantly enhanced cyclase but not hydrolase activity. The differential regulation of CD38 by estradiol-17ß, resulting in increased cADPR synthesis, would have profound effects on calcium regulation and myometrial contractility.

estradiol, female reproductive tract, mechanisms of hormone action, signal transduction, uterus

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Hormones such as estrogens and autacoids influence uterine contractility [1, 2]. The characteristic changes in the pattern and levels of hormones seen during normal estrous cycles, pregnancy, and parturition affect contraction of myometrial smooth muscle through their effect on intracellular calcium ([Ca²⁺]_i) [2, 3]. The regulation of [Ca²⁺]_i in uterine smooth muscle is achieved by several mechanisms. These include activation of phospholipase C by agonists through G-protein-coupled receptors, resulting in formation of inositol 1,4,5-trisphosphate (IP₃) [4–6], influx of Ca²⁺ from the extracellular space via receptor-gated channels [3], and release of Ca²⁺ from intracellular stores [7]. IP₃ causes release of Ca²⁺ from the sarcoplasmic reticulum (SR) through IP₃ receptor channels. In rat myometrium, contractile agonists cause accumulation of inositol phosphates by activating G-protein-coupled receptors [1, 8–11]. Furthermore, the expression of IP₃ receptors and IP₃ binding to its receptors in the uterus are regulated by hormones [12]. In uterine [13] and other smooth muscle [14, 15] cells, SR Ca²⁺ release through activation of ryanodine receptor (RyR) channels also has been shown to have a major role in excitation-contraction coupling. Reverse transcription polymerase chain reaction (RT-PCR) studies have shown that all 3 RyR isoforms are expressed in rat myometrium, and the RyR3 subtype is predominant [13]. In myometrial smooth muscle cells, RyR channels are thought to contribute to Ca²⁺-induced Ca²⁺ release and mechanical activity [16, 17].

Recent studies have shown that cyclic ADP-ribose (cADPR), a metabolite of NAD⁺, mediates Ca²⁺ release from intracellular stores in excitable and nonexcitable cells [18–21]. In airway [22] and vascular smooth muscle [15] cells, studies from our laboratory have demonstrated that cADPR causes Ca²⁺ release through SR RyR channels. Although evidence has been obtained for the regulation of IP₃ production in uterine smooth muscle [23], relatively little is known regarding regulation of cADPR synthesis or degradation in the actions of hormones or autacoids.

The conversion of NAD⁺ to cADPR is catalyzed by the enzyme ADP-ribosyl cyclase and degraded to the inactive analog ADPR by the enzyme cADPR hydrolase [24–26]. In Aplysia ovotestes, ADP-ribosyl cyclase activity is associated with a 29-kDa soluble protein, which has a high degree of similarity to cell surface glycoprotein CD38 of lymphocytes [27]. In a variety of cells, CD38 catalyzes not only the formation of cADPR but also its hydrolysis to ADPR [28]. These results support the concept that CD38 is a protein with bifunctional enzymatic activity. Previous studies have shown that myometrial smooth muscle possesses both ADP-ribosyl cyclase and cADPR hydrolase activities [29]. Regulation of CD38 expression and the kinetics of cADPR formation and degradation would have profound effects on myometrial contractility. In the present study, we investigated whether estradiol-17ß regulates the synthesis of cADPR in myometrium, a target tissue for this hormone, through its genomic effects. We tested the hypothesis that estradiol-17ß regulates cADPR synthesis in the myometrium by increasing CD38 gene expression.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Tris base, Tris HCl, glucose, Hepes, nicotinamide guanine dinucleotide (NGD), cyclic guanosine diphosphoribose (cGDPR), and other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). Hanks balanced salt solution (HBSS), hexamers, oligo(dT), Taq DNA polymerase, and the 100-base pair (bp) DNA ladder were purchased from Gibco BRL (Grand Island, NY). Cellulose polyethyleneimine (PEI) thin-layer chromatography (TLC) plates were purchased from Fisher Scientific (Pittsburgh, PA). Goat polyclonal rat anti-CD38 antibody, donkey anti-goat IgG, and horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Gradient gels and the Bio-Rad protein assay kit were purchased from Bio-Rad Laboratories (Hercules, CA). The estrogen detection kit was purchased from Diagnostic Products Corporation (Los Angeles, CA). Protease inhibitor tablets were obtained from Calbiochem (La Jolla, CA), the RNeasy mini kit was from Qiagen (Valencia, CA), and the SYBR Green Master mix was purchased from Applied Biosystems (Foster City, CA).

Experimental Animals and Design of Studies

Rat myometrial smooth muscle was used in the experiments. Ovariectomized and thyroparathyroidectomized female Sprague-Dawley rats (200–225 g body weight) were obtained from Harlan Laboratories (Madison, WI) and kept in the animal holding facilities for up to 3 wk before the experiments. Rats were given water supplemented with 0.25% calcium gluconate during this period. The 60 animals used in the present study were divided into 2 groups: control (36 rats) and estrogen (E₂) treated (24 rats). Rats in the E₂ group were injected s.c. with estradiol-17ß dissolved in sesame oil at 200 µg kg^-1 day^-1 for 7 days. Control animals received only sesame oil. The animals were killed by i.p. injection of sodium pentobarbital (50 mg/kg) and then exsanguinated. Serum immunoreactive estradiol levels were determined using the commercial kit in an assay standardized and validated as previously described [30]. Using this assay, the serum estradiol levels in control and E₂-treated groups were found to be 55 ± 3.6 pg/ml and 1150 ± 109 pg/ml, respectively. The uterus was removed and kept in ice-cold HBSS buffered with 10 mM Hepes (pH 7.4) and containing 2.5 mM CaCl₂, 1.2 mM MgCl₂, and 10 mM glucose. A small piece of uterus from each rat was removed and frozen immediately in liquid nitrogen for RNA extraction.

Myometrial Smooth Muscle Membrane Preparation

The endometrium and connective tissue from the uteri were removed by dissection. Myometrial tissues were minced with scissors and homogenized using a Polytron tissue homogenizer in 20 mM Tris HCl, pH 7.2, containing 0.25 M sucrose and protease inhibitors (homogenization buffer). The homogenate was centrifuged at 10 000 x g for 15 min to remove cell debris and mitochondria. The postmitochondrial supernatant was centrifuged at 100 000 x g for 60 min, and the pellet (microsomal fraction) was suspended in homogenization buffer. Protein content of the microsomal fractions was determined using the Bio-Rad protein assay kit, with BSA as the standard.

Measurement of ADP-Ribosyl Cyclase and cADPR Hydrolase Activities in Uterine Microsomes

ADP-ribosyl cyclase activity was assayed by measuring the conversion of NGD, an analog of NAD, to cGDPR as an increase in fluorescence intensity at an excitation wavelength of 305 nm and an emission wavelength of 410 nm using an RF-1501 spectrofluorometer (Shimadzu Corporation, Kyoto, Japan) [25]. The fluorescence intensity was converted to nanomoles of cGDPR using a cGDPR standard curve and activity expressed as nmol mg^-1 min^-1. For the assay, approximately 20 µg of microsomal protein was incubated at 37°C in 20 mM Tris HCl buffer, pH 7.2, and the reaction was started by the addition of NGD. Different NGD concentrations (10–400 µM) were used in the assays to determine K_m and V_max values for ADP-ribosyl cyclase using double reciprocal plots.

Cyclic ADPR hydrolase activity in the microsomal fraction was measured using [³²P]cADPR as substrate, as previously described [25]. Approximately 20 µg of microsomal protein was incubated at 37°C in 20 mM Tris HCl buffer, pH 7.2, containing [³²P]cADPR (1200 cpm/µl) and 600 µM unlabeled cADPR. Reactions were initiated by the addition of microsomal protein, and at different times (5–60 min) 1 µl of the reaction mixture was spotted on a PEI cellulose plate. The plate was developed in 0.2 M NaCl in 30% ethanol, dried, and exposed to a phosphorimage screen. The phosphorimage screen was developed using a Cyclone phosphorimager (Packard Instruments, Meriden, CT). The amount of ADPR formed in the reaction was calculated by quantifying the densities of the spots corresponding to cADPR and ADPR, and activity was expressed as pmoles µg^-1 min^-1.

Identification of ADP-Ribosyl Cyclase and cADPR Hydrolase Activities in Uterine Microsomal Proteins Separated by SDS-PAGE or Gel Filtration Chromatography

The proteins in the uterine microsomes were separated by SDS-PAGE. Gels included molecular weight markers. Microsomes from retinoic acid-differentiated HL60 cells, which express CD38, were used as positive controls [31]. Microsomes were loaded and electrophoresed in 12.5% polyacrylamide gels under nonreducing conditions. The gels were rinsed in water 4 times for 15 min and sliced into 5-mm segments with a razor blade. To determine ADP-ribosyl cyclase activity, the slices were incubated in 0.5 ml of 20 mM Tris HCl buffer, pH 7.2, for 24 h at room temperature with 250 µM NGD. An aliquot of the sample was removed, and fluorescence was measured using a fluorescence plate reader. A significant increase in fluorescence above background was due to conversion of NGD to cGDPR. For the determination of cADPR hydrolase activity, the gel slices were incubated for 24 h with 0.3 ml [³²P]cADPR (2500 cpm/µl) containing 200 µM cADPR. After 24 h, a 1-µl aliquot of the solution was removed for determination of cADPR hydrolase activity. Approximate molecular weight of the protein band(s) with the 2 enzyme activities was determined from the following relationship: distance of protein migration/distance of tracking dye migration versus known molecular weight markers.

Uterine microsomal proteins were solubilized using 1% lubrol PX at 4°C for 30 min. Microsomes from rat heart were used as a positive control [25]. Solubilized proteins were recovered by centrifugation at 100 000 x g for 20 minutes and ADP-ribosyl cyclase activity was assayed in the 100 000 x g supernatant to confirm solubilization. The solubilized preparation was applied to a Waters Protein Pak 300SW gel filtration column (8 x 300 mm) along with molecular weight markers using HPLC. The mobile phase consisted of buffer containing 25 mM Hepes (pH 7.4), 250 mM NaCl, and 0.1% lubrol PX. The flow rate was adjusted at 0.5 ml/min. A void volume of approximately 6 ml was determined prior to loading the sample by applying blue dextran (MW 2 000 000) to the column. One-minute fractions (0.5 ml) were collected between 12 and 28 min, and the fractions were assayed for ADP-ribosyl cyclase and cADPR hydrolase activities as described above. An aliquot of the fractions obtained from these columns was subjected to SDS-PAGE separation, followed by Western blot for the detection of CD38.

Western Blot Detection of CD38 in Uterine Microsomes

Uterine microsome (100 µg protein) or the column fractions were electrophoresed on 4–15% polyacrylamide gradient gels, and the proteins in the gels were transferred to a polyvinylidene difluoride (PVDF) membrane. The blots were blocked in PBS containing 1% milk concentrate and 0.025% Tween-20. The PVDF membranes were incubated with a polyclonal goat anti-rat CD38 antibody for 1 h. A horseradish peroxidase-conjugated donkey anti-goat IgG was used as a secondary antibody. The blots were visualized using chemiluminescence substrate before exposure to x-ray film. Standard molecular weight markers were electrophoresed simultaneously for comparing the molecular weights of the visualized proteins in the membrane.

Measurement of CD38 mRNA Expression in Rat Myometrium

Total RNA was extracted using an RNeasy mini kit (Qiagen) as per the manufacturer's protocol. Equal amounts of uterine tissue were taken from the control and E₂-treated rats. Isolated total RNA was quantified using a spectrophotometer, and equal amounts from control and E₂-treated animals were used for RT-PCR. Total RNA was reverse transcribed using random hexamers and oligo(dT) primers to synthesize first strand cDNA from the mRNA. Total RNA (volume adjusted according to the concentration) mixed with random hexamers was heated at 94°C for 5 min. The volume was adjusted to 25 µl using diethyl pyrocarbonate-treated water. RT master mix containing first strand buffer, 0.1 M dithiothreitol, RNase inhibitor, reverse transcriptase (Superscript II), oligo(dT) primer, and distilled water to a final volume of 25 µl was added, and the mixture was incubated at 37°C for 2 h. The first strand cDNA synthesized by RT-PCR was denatured at 94°C for 5 min and amplified by PCR.

The PCR primers were designed using nucleotide sequences for rat CD38 (GenBank accession no. gi 497839). The sense primer was 5'-TGCAACAAGATTCTTCTTTGGAGCA-3' (positions 400–425), and the antisense primer was 5'-CTCAGGATTTTTCACACACTGAAG-3' (positions 876–900), giving a final 500-bp DNA product. The PCR was performed under the following conditions: 94°C for 4 min denaturing, 35 cycles of 94°C for 1 min, 52°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 10 min. The reactions were carried out in a final volume of 50µl in a GeneMate Genius thermocycler (ISC Bioexpress, Kaysville, UT) in the presence of Taq DNA polymerase.

Water was used as a negative control, and mouse GAPDH primers that produced an approximately 500-bp product were used as internal controls. The PCR products were separated on agarose gels and stained with ethidium bromide. The product size was determined by concurrently separating a 100-bp DNA ladder on the gel. The DNA gel was scanned, the intensity of the bands was quantified by densitometry, and results were expressed as the ratio of intensity of CD38 to that of GAPDH in samples from control and E₂-treated rats. The DNA obtained in the myometrial sample was extracted from the gel and sequenced (Advanced Genetic Analysis Center, University of Minnesota, St. Paul, MN) using the forward primer.

Real-Time Quantitative PCR

Quantitative real-time PCR was performed using SYBR Green PCR master mix. Total RNA from control and E₂-treated rat myometrium was converted to first strand cDNA. PCR was carried out in a total volume of 25 µl in the presence of SYBR Green master mix. The CD38 and ß-actin primers were used. The reactions were carried out under the following conditions: 50°C for 2 min, 95°C for 5 min, 40 cycles of 95°C for 45 sec, 50°C for 45 sec, and 72°C for 45 sec, and 1 cycle of 72°C for 10 min. All the reactions were carried out using the ABI Prism 7700 sequence detection system, and the fluorescence was determined 3 times during each cycle. The data were analyzed using Sequence Detection System version 1.7 software. All the samples were run in triplicate, and the readings were normalized using No Template Control and Passive Reference dye included in the SYBR Green Master mix. Normalized fluorescence was plotted against cycle number (amplification plot), and the threshold suggested in the software was used to calculate C_t (cycle at threshold). Results of the real-time PCR were expressed as C_t, and the level of expression of CD38 was indicated by the number of cycles required to achieve the threshold level of amplification. The C_t value from control rats was compared with that of E₂-treated rats.

Statistical Analysis

Data were assessed with a Student t-test for unpaired samples, and significance was set at P < 0.05. Values are presented as mean ± SEM, and n refers to the number of determinations.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of E₂ on ADP-Ribosyl Cyclase and cADPR Hydrolase Activities in Uterine Microsomes

Incubation of uterine microsomes with NGD resulted in time- and NGD concentration-dependent conversion to cGDPR. The conversion of NGD to cGDPR reached a plateau at NGD concentrations of 200 µM (Fig. 1). Uterine microsomes obtained from E₂-treated rats exhibited increased rates of conversion of NGD to cGDPR (Fig. 2A) and significantly higher specific activity (Fig. 2B; 6.04 ± 1.3 vs. 10.9 ± 1.9 nmoles mg^-1 min^-1 in control and E₂-treated groups, respectively; P < 0.05). For the different microsomal preparations, a double reciprocal plot was generated and used to calculate V_max and K_m values for ADP-ribosyl cyclase activity (Fig. 3). The K_m values for ADP-ribosyl cyclase activity in microsomes from control and E₂-treated rats calculated from these plots were 50.3 ± 15.9 µM and 45.4 ± 9.7 µM, respectively. Similarly, the V_max values for ADP-ribosyl cyclase activity in microsomes from control and E₂-treated rats calculated from the double reciprocal plots were 11.8 ± 3.7 nmoles mg^-1 min^-1 and 10.2 ± 3 nmoles mg^-1 min^-1, respectively. The K_m and V_max values for control and E₂-treated groups were not significantly different (P > 0.05).

View larger version (19K): [in this window] [in a new window]   FIG. 1. ADP-ribosyl cyclase activity in rat uterine microsomes. Microsomes (20 µg of microsomal protein) were incubated with different concentrations of NGD (10–400 µM), and the fluorescence intensity was measured over time. Tracing shows mean ± SEM velocity (nmoles mg-1 min-1) versus NGD concentration. The activity reaches a plateau at an NGD concentration of 200 µM (n = 6)

View larger version (19K): [in this window] [in a new window]   FIG. 2. ADP-ribosyl cyclase activity in microsomes obtained from control and E2-treated rats. A) Representative tracing of velocity of ADP-ribosyl cyclase activity (expressed in fluorescence units) versus time (in seconds) in the presence of 200 µM NGD and 20 µg of microsomal protein. B) Specific activity of ADP-ribosyl cyclase (nmoles mg-1 min-1) in uterine microsomes obtained from control (n = 14) and E2-treated (n = 16) rats. Values are mean ± SEM. *Significantly different from control (P < 0.05)

View larger version (13K): [in this window] [in a new window]   FIG. 3. A representative double reciprocal plot of ADP-ribosyl cyclase activity versus NGD concentration in microsomes obtained from control rats (A) and E2-treated rats (B). Km and Vmax values were calculated from such plots (n = 4)

Incubation of uterine microsomes with [³²P]cADPR resulted in conversion to [³²P]ADPR, as shown in the phosphorimage of a representative TLC plate (Fig. 4A). The cADPR hydrolase activities in microsomes from control and E₂-treated rats, measured at different time periods, did not differ significantly (P > 0.05) (Fig. 4B). The specific activity of cADPR hydrolase in uterine microsomes from control and E₂-treated rats, calculated from the initial velocities, did not differ significantly (Fig. 4C). Cytosolic fractions obtained from myometrial smooth muscle of control or E₂-treated rats had no measurable ADP-ribosyl cyclase or cADPR hydrolase activities (data not shown).

View larger version (24K): [in this window] [in a new window]   FIG. 4. A) Representative phosphorimage of a TLC plate showing conversion of [32P]cADPR to [32P]ADPR, measured at 3 time points (20, 30, and 60 min). Spots at the bottom of the image reflect ADPR formed, and those at the top reflect cADPR remaining. Lanes 1–5 for each time period represent different microsomal preparations. Lane 6 for each time period is the control, i.e., in the absence of microsomes, where there is no significant conversion of [32P]cADPR to [32P]ADPR. Data represent 7–13 separate determinations. B) Representative plot of percentage of cADPR hydrolyzed versus time in the presence of uterine microsomes (20 µg protein) from control and E2-treated rats. C) Velocity of cADPR hydrolase activity in microsomes obtained from control and E2-treated rats, measured after 20 min incubation. There is no significant difference in cADPR hydrolase activities in microsomal preparations between control and E2-treated rats at this or any of the time periods. Velocities (pmoles µg-1 min-1) are represented as mean ± SEM (n = 7–11 for control and 9–13 for E2 treated)

Western Blot Detection of CD38 in Uterine Microsomes

Uterine microsomal proteins were fractionated by SDS-PAGE, and the separated proteins were transferred to a PVDF membrane and probed with an anti-CD38 antibody in the Western blots. CD38 expression was significantly greater in uterine microsomes from E₂-treated rats than in those from controls (Fig. 5). Densitometric analysis of the bands in the Western blots revealed a 2.7-fold increase in CD38 expression in microsomes from E₂-treated rats as compared with those from controls.

View larger version (61K): [in this window] [in a new window]   FIG. 5. Representative Western blot showing increased CD38 expression in uterine microsomes from E2-treated rats compared with controls. Lane 1 represents molecular weight markers, lanes 2 and 3 are uterine microsomes from 2 different control rats, and lanes 4 and 5 are uterine microsomes from 2 different E2-treated rats. Arrows indicate the CD38 band at a molecular weight of 45 000. Data represent 3 separate determinations.

Analysis of CD38 Expression by RT-PCR and Real-Time Quantitative PCR

Total RNA isolated from uterine tissues was reverse transcribed to cDNA and further amplified using primers specific for rat CD38. The PCR amplification products were separated on agarose gels and stained with ethidium bromide. In myometrium from E₂-treated animals, there was a 100-fold increase in total RNA content as compared with controls. The intensity of the bands in control and E₂-treated myometrial samples was compared to assess the genomic effect of estrogen on CD38 expression. The nucleotide sequence of the 500-bp PCR product was analyzed and confirmed to be that of rat CD38. There was enhanced CD38 mRNA content in myometrium obtained from E₂-treated rats as compared with controls (Fig. 6). Ratiometric analysis of the intensity of the bands showed an approximately 2-fold increase (0.275 vs. 0.143) in CD38 content in myometrial samples from E₂-treated rats.

View larger version (48K): [in this window] [in a new window]   FIG. 6. Representative gel showing CD38 mRNA expression in rat myometrium by RT-PCR analysis. Lanes 1 and 7 represent 100-bp DNA ladder (600-bp band indicated by arrows), lanes 2 and 3 represent PCR products using GAPDH primers from control and E2-treated rats, respectively, and lanes 4 and 5 represent PCR products using rat CD38 primers from control and E2-treated rats, respectively. Note increased expression of CD38 (500-bp product in the gel) in the sample from the E2-treated rat (Trt) as compared with the control rat (Cntr). Lane 6 represents PCR product using sterile water as a template with rat CD38 primers. Data represent 3 separate determinations

Real-time PCR of RNA extracted from myometrium of control and E₂-treated rats confirmed increased expression of CD38. The mean C_t values for control and E₂-treated samples were 27.6 and 26.9, respectively.

ADP-Ribosyl Cyclase and cADPR Hydrolase Activities in the Microsomal Fraction Separated by Gel Filtration Chromatography or SDS-PAGE

Comigration of ADP-ribosyl cyclase and cADPR hydrolase was confirmed using gel filtration chromatography (Fig. 7). Both enzyme activities were contained in the same protein fractions collected from the gel filtration column, with a molecular weight of 90 kDa representing a CD38 oligomer, most likely a dimer. Western blot analysis of the column fractions showed the highest CD38 immunoreactivity in the same fractions that had the highest ADP-ribosyl cyclase and cADPR hydrolase activities, i.e., fractions 5–8 (Fig. 7). Comigration of ADP-ribosyl cyclase and cADPR hydrolase was also confirmed by SDS-PAGE separation of microsomal proteins. Both enzymes in the uterine microsomes comigrated in the same gel slice, at a molecular weight of 45 kDa (determined from position of molecular weight markers in the gel; data not shown). In microsomes obtained from retinoic acid-stimulated HL60 cells and rat heart separated by either gel filtration chromatography or SDS-PAGE, ADP-ribosyl cyclase and cADPR hydrolase comigrated in the same fractions as in uterine microsomes (data not shown).

View larger version (25K): [in this window] [in a new window]   FIG. 7. Comigration of ADP-ribosyl cyclase (A) and cADPR hydrolase (B) in uterine microsomes from control and E2-treated rats fractionated by gel filtration chromatography. Both enzymes in uterine microsomes peak at the same column fraction with a protein of 90 000 molecular weight, representing an apparent dimer. Western blot (C) of column fractions 4–9 shows the presence of 45 000 molecular weight CD38 in fractions 5–8 (n = 3)

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study, we demonstrated that estradiol-17ß increases CD38 expression in rat myometrium. The increased CD38 expression is reflected by significantly enhanced ADP-ribosyl cyclase activity in uterine microsomes. SDS-PAGE and gel filtration chromatography results indicate that the ADP-ribosyl cyclase and cADPR hydrolase comigrate with a single protein with molecular weight similar to that of CD38. Analysis of cADPR hydrolase activity in uterine microsomes from E₂-treated and ovariectomized rats revealed no significant difference. The differential effect of estradiol-17ß on ADP-ribosyl cyclase and cADPR hydrolase activities in the myometrium would favor increased synthesis of cADPR during agonist stimulation.

The role of cADPR in Ca²⁺-induced Ca²⁺ release has been demonstrated in a variety of cell types [15, 18–22]. It is widely believed that NAD is the physiological ligand for CD38 [32], a transmembrane type II glycoprotein. CD38 is one of the NAD glycohydrolases (NADase) and is a bifunctional ectoenzyme [33, 34]. It catalyzes the formation and hydrolysis of cADPR and thus possesses both ADP-ribosyl cyclase and cADPR hydrolase activities. Regulated expression of CD38 in cells is expected to have profound effects on cADPR synthesis and therefore intracellular Ca²⁺ regulation.

Recent studies have shown that hormones and neurotransmitters increase levels of cADPR in a variety of cell types [35–37]. It is not clear, however, whether this increase results from activation of ADP-ribosyl cyclase through G-protein-coupled receptors or whether regulation is downstream to receptors and is achieved by second messengers. In this context, studies have indicated that nitric oxide and cGMP augment cADPR production [38], suggesting the possibility that cGMP-dependent phosphorylation of CD38 regulates ADP-ribosyl cyclase activity. In adrenal chromaffin cells [36] and NG108-15 neuronal cells [39], ADP-ribosyl cyclase activity appears to be regulated via G proteins that are activated by muscarinic receptors. In cardiac myocytes, cADPR synthesis is stimulated by activation of ADP-ribosyl cyclase via G-protein-coupled ß-adrenoceptors [35]. Other studies have provided evidence for regulation of ADP-ribosyl cyclase activity in vascular smooth muscle by hormones and other stimuli [40]. In a recent study, Chini et al. [29] presented evidence for estrogen stimulation of ADP-ribosyl cyclase and cADPR hydrolase activities in rat myometrium. The increased ADP-ribosyl cyclase activity was attributed to higher V_max value, with no significant change in K_m. Estrogen effects were blocked by the estrogen antagonist tamoxifen, suggesting actions mediated through estrogen receptors. Although our results on stimulation of ADP-ribosyl cyclase activity confirm those of Chini et al. [29], the differential effects of estrogen on ADP-ribosyl cyclase and cADPR hydrolase activities reported in the present study are novel, as are the genomic effects of estrogen on CD38 expression in the myometrium.

In smooth muscle, there is very little information concerning the subcellular localization of ADP-ribosyl cyclase and cADPR hydrolase activities. In a recent study, we provided evidence for localization of ADP-ribosyl cyclase and cADPR hydrolase activities in airway smooth muscle plasma membrane, with little if any activity in the cytosolic fraction [25]. Uterine microsomal fractions separated by SDS-PAGE or gel filtration showed comigration of ADP-ribosyl cyclase and cADPR hydrolase with a single protein fraction, suggesting that the enzyme activities are attributable to a single bifunctional protein. Western blot analysis of uterine microsomes confirms that this protein is CD38, whose expression is significantly augmented by treatment with estradiol-17ß. The increased ADP-ribosyl cyclase activity is most probably due to increased CD38 content in the uterine microsomes and/or the result of a stable modification of the enzyme during estrogen treatment.

Because CD38 is an ectoenzyme with bifunctional activity, increased CD38 expression induced by estrogen is expected to result in upregulation of both enzyme activities. In the present study, estrogen caused upregulation of ADP-ribosyl cyclase activity but not cADPR hydrolase activity. Although the mechanisms underlying this differential regulation are not known, it most likely stems from some posttranslational modification of CD38. Estrogen may upregulate pathways or mechanisms that can lead to selective posttranslational modification of critical amino acid residues contributing to ADP-ribosyl cyclase activity. The role of critical amino acid residues in CD38 contributing to enzyme activity has been investigated. Recent studies have shown that lymphocyte ectoenzymes regulate lymphocyte function through their selective effects on CD38 [41]. ADP-ribosylation of arginine residues in CD38 by ecto-mono-ADP-ribosyltransferases leads to inactivation of both ADP-ribosyl cyclase and cADPR hydrolase activities, whereas that of cysteine residues results in inhibition of cADPR hydrolase but not ADP-ribosyl cyclase activity [42]. These findings were further confirmed by site-directed mutagenesis of arginine or cysteine residues in the CD38 and measurement of the functional consequence of such changes. Posttranslational modification through phosphorylation of specific amino acid residues in a protein with bifunctional enzyme activity resulting in differential regulation has also been described [43]. Estrogen may cause selective modulation of CD38 through its effects on pathways such as ADP-ribosylation or phosphorylation by different kinases, which selectively augment ADP-ribosyl cyclase activity. Other potential mechanisms for differential regulation by estrogen include an mRNA splice variant of CD38 encoding a different isoform. Although evidence for the presence of an alternative splicing isoform of CD38 has been presented, the expressed protein appears to lack both ADP-ribosyl cyclase and cADPR hydrolase activities [44]. Other reports have indicated presence of hormone-responsive motifs in human CD38 promoter sites, suggesting the possibility for estrogen regulation of CD38 expression [45].

Estrogens increase uterine contractility through their effects on intracellular Ca²⁺ regulation [2]. Our findings support the hypothesis that estrogen regulates uterine contractility by increasing CD38 expression and ADP-ribosyl cyclase activity and by decreasing cADPR hydrolase activity. The net effect of differential regulation of the enzymes involved in the synthesis and degradation of cADPR by estrogen would favor increased cADPR production during agonist stimulation, as indicated by the increased rate of conversion of NGD to cGDPR by microsomes obtained from E₂-treated rats. These changes would have a profound effect on myometrial motility during normal estrous cycles or pregnancy.

	FOOTNOTES

 
First decision: 16 July 2001.

¹ This work was supported by grants from the National Institutes of Health (HL057498 to M.S.K. and DA11806 to T.F.W.), an Academic Health Center Faculty Development Grant, University of Minnesota, and a Fellowship from the Turkish Government (S.D.).

² Correspondence: Mathur S. Kannan, Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Ave., St. Paul, MN 55108. FAX: 612 625 5203; kanna001{at}tc.umn.edu

Accepted: October 4, 2001.

Received: June 11, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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