Modulation of nerve growth factor in peripheral organs by estrogen and progesterone

PII: S 0 3 0 6 - 4 5 2 2 ( 0 1 ) 0 0 5 6 8 - 1

Neuroscience Vol. 110, No. 1, pp. 155^167, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00

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MODULATION OF NERVE GROWTH FACTOR IN PERIPHERAL ORGANS BY ESTROGEN AND PROGESTERONE D. E. BJORLING,a;b * M. BECKMAN,c M. K. CLAYTONd and Z.-Y. WANGa a

Department of Surgical Sciences, School of Veterinary Medicine, The University of Wisconsin, 2015 Linden Drive, West, Madison, WI 53706, USA b

Division of Urology, Department of Surgery, School of Medicine, The University of Wisconsin, 600 Highland Avenue, Madison, WI 53792-3236, USA c

Orthopaedic Research Laboratory, 1112 East Clay Street, P.O. Box 980694, Virginia Commonwealth University, Richmond, VA 23298-0694, USA d

Department of Statistics, The University of Wisconsin, 1210 Dayton Street, Madison, WI 53706-1685, USA

AbstractöNerve growth factor (NGF) synthesized in peripheral organs plays a critical role in the development and maintenance of the nervous system and also participates in processing nociceptive stimuli. Previous studies suggest that reproductive hormones may regulate the expression of NGF. Ovariectomies were performed on female mice, and mice were killed 24 h after hormone replacement to evaluate the e¡ects of estrogen and progesterone on NGF in peripheral organs, speci¢cally the uterus, bladder, heart, and salivary gland. Sham-operated intact mice and untreated ovariectomized mice served as controls. Immunohistochemistry demonstrated the presence of NGF, estrogen receptor-K, estrogen receptor-L, and progesterone receptors in these organs. Ovariectomy caused a signi¢cant decrease in NGF protein content in the uterus, and short term treatment of ovariectomized mice with estrogen and/or progesterone increased uterine NGF mRNA and restored NGF protein to concentrations similar to intact control mice. Ovariectomy did not a¡ect NGF protein concentrations in the salivary gland, but treatment of ovariectomized mice with estrogen alone or in conjunction with progesterone stimulated concentrations of NGF protein that exceeded those observed in intact control or ovariectomized, untreated mice. NGF mRNA was increased in salivary glands from ovariectomized mice treated with progesterone alone or in combination with estrogen relative to other groups. NGF protein content of the hearts of ovariectomized mice treated with estrogen alone or in conjunction with progesterone was increased relative to intact controls and ovariectomized, untreated mice, but neither ovariectomy or hormone replacement a¡ected NGF mRNA content in the heart. NGF protein content of the bladder was una¡ected by ovariectomy or hormone treatment, and bladder NGF mRNA was una¡ected by ovariectomy or hormone treatment. Collectively, these results indicate that reproductive hormones have the capacity to regulate NGF message and protein in a manner that varies among organs. Fluctuations in the expression of NGF, in conjunction with other factors, may help to explain gender di¡erences in pain sensation and in£ammatory response. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: mice, ovariectomy, uterus, bladder, heart, salivary gland.

ity in adults (McAllister et al., 1999; Scully and Otten, 1995). NGF may also play a critical role in nociception, because injection of NGF into humans causes immediate and prolonged pain (Petty et al., 1994). NGF produced in peripheral tissues is internalized by sympathetic and small sensory nerve ¢bers through binding with the high a¤nity tyrosine kinase A receptor (trkA) and transported in a retrograde direction to the dorsal root ganglia where it stimulates neuronal survival via at least three separate cell signaling pathways initiated by Ras activation (Kaplan and Miller, 2000). Retrograde transport of NGF to dorsal root ganglia also appears to initiate transcriptional changes that in£uence central events relevant to nociception (Lindsay and Harmar, 1989; Longo et al., 1993; Vedder et al., 1993). Interactions between NGF and estrogen in the cerebral cortex, basal forebrain, and the dorsal root ganglion are well documented in adult animals (Gibbs, 1998; Sohrabji et al., 1994b). The concept that estrogen may

Nerve growth factor (NGF) is a trophic factor that is essential for development and continued function of various cell types within the CNS and sympathetic and sensory neurons of the peripheral nervous system (Davies, 1994; Elkabes et al., 1996; Longo et al., 1993). Production of NGF and other neurotrophins in peripheral (`target') organs stimulates prenatal development of intrinsic innervation and also appears to modulate neural plastic-

*Corresponding author. Tel.: +1-608-2634808; fax: +1-6082637930. E-mail address: [email protected] (D. E. Bjorling). Abbreviations : ANOVA, analysis of variance; ELISA, enzymelinked immunosorbent assay; ER, estrogen receptor; NGF, nerve growth factor; PCR, polymerase chain reaction ; PR, progesterone receptor ; RT, reverse transcription; trkA, tyrosine kinase A receptor. 155

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regulate expression and the biological e¡ects of NGF is reinforced by the observation that motifs that mimic a classical estrogen response element are found in the promoter and 5P-£anking regions of the genes for human NGF, p75 (the low a¤nity receptor for NGF), and trkA (Sohrabji et al., 1994a, b; Toran-Allerand et al., 1992; Toran-Allerand, 1996). It has been observed that estrogen receptors (ERs) and trkA are co-localized in a sub-population of dorsal root ganglion neurons, and estrogen up-regulates expression of trkA in sensory neurons in a concentration-dependent manner (Sohrabji et al., 1994b). Concentrations of trkA mRNA in the forebrain of female rats were found to £uctuate during the estrus cycle and were generally lowest on the afternoon of proestrus (coinciding with the peak plasma estrogen concentration) and highest during estrus and the ¢rst day of diestrus (Gibbs, 1998). Less is known about regulation of NGF content in peripheral organs by steroidal hormones. The density of sympathetic innervation of the uterus is decreased during pregnancy, and there is a paucity of sympathetic nerve ¢bers innervating the uterus in late pregnancy (Haase et al., 1997; Marshall, 1981; Moustafa, 1988). It has been suggested that this may contribute to maintenance of pregnancy by suppressing motor function of the uterus (Varol et al., 2000). Interstitial cystitis is a painful bladder disorder of uncertain etiology that a¡ects predominantly women (Held et al., 1990). Epidemiologic studies indicate that the severity of interstitial cystitis symptoms wax and wane in some female patients as circulating concentrations of reproductive hormones £uctuate; as many as 40% of interstitial cystitis patients report that their symptoms worsen perimenstrually (particularly around the time of ovulation), and many interstitial cystitis patients ¢nd that symptoms improve during pregnancy (Held et al., 1990; Ratner et al., 1992; Whitmore, 1994). Increased NGF was observed in the mucosa of bladder biopsies obtained from female patients with painful bladder disorders (including interstitial cystitis) (Lowe et al., 1997), and concentrations of NGF, neurotrophin-3, and glial-derived neurotrophin factor were increased in urine obtained from patients with interstitial cystitis (Okragly et al., 1999). These observations suggest that estrogen and/or progesterone may regulate NGF expression in peripheral organs, and that £uctuations in NGF in these organs in response to steroidal hormones may play a critical role in neural plasticity and nociception. NGF protein and mRNA were measured in the uterus, bladder, heart, and salivary gland in intact female mice (no treatment) and in ovariectomized mice that received hormone supplementation to test the hypothesis that estrogen and/or progesterone modulate NGF in these organs. The salivary gland and heart were included in these investigations for comparison to organs within the urogenital tract because both organs express estrogen and progesterone receptors (PRs), NGF was ¢rst isolated from the salivary glands (Shooter, 2001) and the salivary glands are a rich source of a variety of growth factors (Jaskoll and Melnick, 1999), and the heart is a major organ in a separate body cavity.

EXPERIMENTAL PROCEDURES

Animals Female Balb/c mice (20 g, 10^12 weeks old) were obtained from Harlan (Indianapolis, IN, USA) and used in accordance with an animal care protocol approved by the University of Wisconsin Institutional Animal Care and Use Committee. The minimal number of animals necessary to satisfy statistical signi¢cance was used, and analgesics were administered subsequent to surgical procedures. When it was necessary to determine the estrus phase of an animal, this was done by examining vaginal cytology according to standardized criteria (Bronson et al., 1975). Surgery Mice were anesthetized with a single intramuscular injection of ketamine HCl (100 mg/kg) and xylazine (12 mg/kg). The skin overlying the thoracolumbar spine was prepared for aseptic surgery, and the ovaries were removed through a single dorsal midline incision in the skin and bilateral incisions in the dorsal £ank. In sham-operated mice, the ovaries were inspected, and the wounds were closed. Ovariectomized mice were killed 2 weeks after surgery, and sham-operated control mice were killed at least 2 weeks after surgery when they were determined to be in proestrus by vaginal swabbing. Hormone treatment Two weeks after surgery, ovariectomized animals were divided into four groups: control animals given a single subcutaneous injection of sesame oil (0.1 ml) 24 h prior to being killed; estrogen-treated mice that received a single injection of 17-L-estradiol (250 ng in sesame oil, 0.1 ml total volume; Sigma, St. Louis, MO, USA) 24 h prior to being killed; progesteronetreated mice that received daily subcutaneous injections of progesterone (1 mg in sesame oil, 0.1 ml total volume; Sigma) given once daily for four consecutive days prior to being killed; and mice treated with a combination of estrogen and progesterone (i.e. four daily injections of progesterone and a single injection of estrogen given concurrently to the fourth injection of progesterone 24 h prior to being killed). Although the dose of estrogen used may be considered supraphysiologic (Acu¡ et al., 1997), the doses and timing of combined administration of estrogen and progesterone that were used are considered to mimic the early phases of pregnancy (Wang et al., 1994). Further, these doses of hormones have been shown to alter mRNA and associated protein content of heparin-binding epidermal growth factor, Muc-1 mucin, and C-type natriuretic peptide in the uterus of the ovariectomized mouse (Acu¡ et al., 1997; Braga and Gendler, 1993; Wang, 1994). Tissue harvesting Mice were deeply anesthetized with methoxy£urane, and the uterus, bladder, heart, and submandibular salivary glands were removed. Tissues used for analysis of NGF protein content were weighed immediately, homogenized in the presence of proteinase inhibitors, and stored at 370³C to allow enzyme-linked immunosorbent assays (ELISAs) to be performed concurrently, and tissues used for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) were placed immediately in RNAlater0 solution (Ambion, Austin, TX, USA) and stored at 4³C. Immunohistochemistry Intact mice determined to be in proestrus were anesthetized as described above and perfused with heparinized saline, followed by 4% paraformaldehyde (40 ml, pH 7.4) at a rate of 10 ml/min. Uteri, bladders, hearts, and salivary glands were removed and post-¢xed in 4% paraformaldehyde for an additional 4 h at 4³C.

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Tissues were cryoprotected with 20% sucrose and frozen in embedding solution on dry ice. Sections (10 Wm) were cut using a cryostat (Microm Laborgerate, Walldorf, Germany) and mounted onto gelatin-treated slides. Transverse sections were obtained from the middle of the uterine horns (halfway between the tubal end and the bifurcation), the body of the bladder, and the long axis of the heart and salivary gland. Sections were air-dried, rinsed in phosphate-bu¡ered saline several times, and incubated with 3% H2 O2 for 10 min to quench endogenous peroxidase activity. Tissues were then treated with 10% normal goat serum for 2 h at room temperature in a humid chamber. Sections were incubated overnight at 4³C with each speci¢c antibody. Staining was revealed using a TSA1 Fluorescence System (NEN Life Science Products, Boston, MA, USA) following the manufacturer's instructions. This system uses tyramide reagents to amplify the staining signals. Sections were rinsed and coverslipped using an anti-fading solution (Vectashield, Vector laboratories, Burlingame, CA, USA). Sections were examined with a Nikon E600 microscope, and digital images were captured using a digital camera (Diagnostic Instruments, Sterling Heights, MI, USA). For negative controls, tissue sections were incubated with normal rabbit IgG instead of speci¢c antibodies. The following antibodies (all raised in rabbits) were used: anti-NGF (1:500); anti-estrogen receptor-K (ERK; 1:2000); anti-estrogen receptor-L (ERL; 1:1000); and anti-progesterone receptor (PR; 1:3000). The anti-NGF antibody was obtained from Chemicon (Temecula, CA, USA), and all other antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NGF protein content of tissues NGF protein content of organs was determined by ELISA. The entire uterine horns and body (up to, but not including, the cervix), bladder, salivary gland, and heart (after removal of adjacent fat and the great vessels) were used. Tissues were blotted, weighed, and homogenized in 0.5 ml ice-cold extraction medium of the following composition: Tris^HCl (100 mM), NaCl (0.4 M), sodium azide (0.05%), Triton X-100 (1%), phenylmethylsulphonyl £uoride (1 mM), EDTA (4 mM), aprotinin (0.09 TIU/ml), pH 7.0. Samples were treated with 1 M HCl (1 Wl/ 50 Wl sample) for 15 min at room temperature and then neutralized by adding 1 M NaOH (1 Wl/50 Wl sample). The pH of samples was checked and, if necessary, adjusted to pH 7.6. After homogenization, samples were spun at 3000 rpm for 15 min at 0³C. Tissue supernatants were frozen at 370³C until analysis by ELISA. Homogenates from uteri, bladders, and hearts were assayed at a 1:10 dilution, whereas homogenates from submandibular glands were evaluated at 1:32 000 to 1:64 000 dilutions. The Nerve Growth Factor Emax ImmunoAssay System0 (Promega, Madison, WI, USA) was used. Ninetysix-well plates were coated with goat anti-NGF polyclonal antibody that binds soluble NGF protein in the extracts. Captured NGF was detected by a second anti-NGF monoclonal antibody (rat origin), and the amount of speci¢cally bound monoclonal antibody was measured. This assay is accurate for NGF protein content between 7.8 and 500 pg/ml with less than 2% crossreactivity with other neurotrophins at 10 ng/ml. Assays were run in duplicate and were repeated if greater than 10% di¡erence was observed between replicates. Protein content of the samples was determined using the Bio-Rad Protein Assay0 ; (Bio-Rad Laboratories, Hercules, CA, USA), and NGF protein was normalized to total protein content. Recovery of NGF protein from samples was evaluated by adding recombinant human NGF (Genentech, South San Francisco, CA, USA) to samples of bladder tissue in concentrations ranging from 20 to 200 pg NGF/mg protein. Recovery of known concentrations of NGF protein consistently ranged from 89 to 92%, and regression of recovery against known quantities added produced a value of r = 0.931. Semi-quantitative RT-PCR Total RNA was extracted using the RNeasy Mini Kit0 ; (Qia-

157

gen, Valencia, CA, USA) and reverse-transcripted to cDNA. To quantify NGF mRNA expression levels, PCR was done using a PE Biosystems Model 5700 TaqMan0 ; instrument (Morrison et al., 1998; Wittwer et al., 1997). Basically, this instrument monitors real-time PCR product accumulation by using SYBR Green, a dye that £uoresces upon binding to double-stranded DNA. Serial dilutions of cDNA sample were prepared, and the relative concentrations of these dilutions were plotted against the PCR cycle number at which PCR product accumulation reached a de¢ned threshold (as re£ected by £uorescence measurements). The resulting standard curve was used to determine relative concentrations of NGF mRNA in each sample. To normalize for the amount of input RNA in the cDNA synthesis reaction, a similar method was used to establish the relative concentrations of message for L19, a ribosomal protein. All samples were run in duplicate, and the expression levels of NGF mRNA were normalized to L19 mRNA in each sample. Primer sequences used for NGF were: 5P-GCC AAG GAC GCA GCT TTC TAT (forward) and 5P-CGC AGT GAT CAG AGT GTA GAA CAA C (reverse). Those used for L19 were: 5P-CTG AAG GTC AAA GGG AAT GTG (forward) and 5P-GGA CAG AGT CTT GAT GAT CTC (reverse). Data analysis Protein and mRNA values did not follow a normal distribution, and hence non-parametric analyses were used. Speci¢cally, for each tissue and each outcome measure (protein and mRNA), observations were ranked using the RANK procedure of SAS (SAS Institute, Cary, NC, USA) and the ranked values were then analyzed as a one-way analysis of variance (ANOVA) using the GLM procedure of SAS. When the overall F-test in the ANOVA was signi¢cant (P 6 0.05), mean ranks were compared using the least squares di¡erence method. All data is presented as mean þ S.E.M.

RESULTS

E¡ect of ovariectomy and hormone replacement on uterine weight The weight of the uterine horns and body were signi¢cantly less in ovariectomized (12.1 þ 4.6 mg/20 g body weight) than sham-operated intact (26 þ 5.3 mg/20 g body weight) mice. Treatment of ovariectomized mice with progesterone did not increase uterine weight (15.4 þ 3.9 mg/20 g body weight), but there was no di¡erence in uterine weight in ovariectomized mice treated with estrogen (24.2 þ 5.8 mg/20 g body weight) and intact sham-operated intact mice. Treatment of ovariectomized mice with estrogen and progesterone stimulated a signi¢cant increase in uterine weight (39.6 þ 6.3 mg/20 g body weight). Immunohistochemistry Moderate staining of NGF protein was observed in the epithelial cells of mucosa of the uterus (Fig. 1). More di¡use staining was occasionally observed in the endometrial stroma as well as in the myometrium. Moderate to strong staining of ERK and ERL was present in the epithelium and some glands of the uterus. Staining of PR occurred predominantly in stroma cells with occasional staining observed in glandular cells. The secretory cells of the salivary gland stained very strongly for NGF protein, and moderate staining for

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Fig. 1. Immunohistochemistry of the uterus. Moderate staining of NGF was observed in the epithelial cells of mucosa (arrows) and di¡use staining was observed in the endometrial stroma as well as in the myometrium. Staining of both ER-K and -L occurred in the epithelial layer (arrows) and in some glands, although staining of ER-K was somewhat stronger than that of ER-L. Strong staining of PR was mainly observed in the stroma, and some glandular cells also showed moderate staining of PR. Speci¢c staining was not observed when tissues were exposed to normal rabbit IgG instead of speci¢c antibodies. Scale bar = 50 Wm.

ERK and -L was also observed in secretory cells (Fig. 2). Staining for PR occurred predominantly in cells lining the ducts of the salivary gland, but weaker staining was also observed in secretory cells.

Weak and di¡use staining of NGF protein, ERL, and PR was observed in the cardiac muscle (Fig. 3). Strong staining of ERK was present in the nuclei of cardiac muscle cells.

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159

Fig. 2. Immunohistochemistry of the salivary gland. Very strong staining of NGF was seen in many secretory cells, and moderate staining of ER-K and -L was also observed in secretory cells. Moderate staining of PR occurred in the duct cells, and weaker staining was observed in secretory cells as well. Speci¢c staining was not observed when tissues were exposed to normal rabbit IgG instead of speci¢c antibodies. Scale bar = 50 Wm.

Staining for NGF protein, ERK, ERL, and PR occurred primarily in the mucosa of bladder (Fig. 4). Weak di¡use staining was occasionally observed in the detrusor (muscle).

Expression of NGF The e¡ects of ovariectomy and hormone replacement on NGF protein content and mRNA abundance varied

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Fig. 3. Immunohistochemistry of the heart. Weak and di¡use staining of NGF, ER-L, and PR was observed in cardiac myocytes. Strong staining of ER-K was observed in the nuclei of cardiac myocytes. Speci¢c staining was not observed when tissues were exposed to normal rabbit IgG instead of speci¢c antibodies. Scale bar = 50 Wm.

among organs (Tables 1 and 2). Ovariectomy stimulated a signi¢cant decrease in NGF protein in the uterus (Table 1). Treatment of ovariectomized mice with estrogen or progesterone alone or in combination resulted in a signi¢cant increase in NGF protein relative to concentrations observed in uteri from ovariectomized mice, but

concentrations of uterine NGF protein in ovariectomized mice treated with hormones were not signi¢cantly di¡erent from those observed in intact sham-operated controls. Ovariectomy caused a signi¢cant decrease in NGF mRNA in the uterus, and treatment of ovariectomized mice with estrogen caused a signi¢cant increase in

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161

Fig. 4. Immunohistochemistry of the bladder. Moderate staining of NGF and ER-L was observed in the mucosa of the bladder (arrows), and weak, di¡use staining for NGF and ER-L occurred in the detrusor (muscle). Strong staining of ER-K and PR was present in the mucosa. Speci¢c staining was not observed when tissues were exposed to normal rabbit IgG instead of speci¢c antibodies. Scale bar = 50 Wm.

uterine NGF mRNA relative to that observed in ovariectomized mice or intact controls (Table 2). Treatment of ovariectomized mice with progesterone resulted in uterine NGF mRNA content that was greater than that observed in intact sham-operated controls, ovariecto-

mized, or ovariectomized mice treated with estrogen, and treatment of ovariectomized mice with estrogen and progesterone stimulated concentrations of uterine mRNA that exceeded those observed in any of the other groups.

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D. E. Bjorling et al. Table 1. NGF protein content in tissues

Uterus (pg/mg protein)

Salivary gland (Wg/mg protein)

Heart (pg/mg protein)

Bladder (pg/mg protein)

Treatment

n

Mean þ S.E.M.

sham OVX OVX+E2 OVX+PG OVX+E2+PG sham OVX OVX+E2 OVX+PG OVX+E2+PG sham OVX OVX+E2 OVX+PG OVX+E2+PG sham OVX OVX+E2 OVX+PG OVX+E2+PG

12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12

480.8 þ 116.0 165.3 þ 7.2 1121.8 þ 383.2 494.7 þ 29.0 747.6 þ 186.1 0.54 þ 0.07 0.41 þ 0.1 1.40 þ 0.27 1.37 þ 0.36 1.61 þ 0.28 786.0 þ 99.6 798.7 þ 35.1 999.4 þ 67.6 950.3 þ 73.0 1099.1 þ 64.0 106.0 þ 42.0 76.8 þ 15.2 113.8 þ 30.7 79.5 þ 15.2 69.4 þ 15.4

Change (%)a

Change (%)b

P*

P**

365.5 +133.3 +2.9 +55.5

+578.6 +199.3 +352.3

6 0.001 ns ns ns

6 0.001 6 0.001 6 0.001

331.7 +159.2 +153.7 +198.1

+241.5 +234.1 +292.7

ns 6 0.001 ns 6 0.001

6 0.001 6 0.001 6 0.001

+1.6 +27.2 +20.9 +39.8

+25.1 +19.0 +37.6

ns 6 0.001 ns 6 0.001

6 0.001 6 0.001 6 0.001

327.5 +7.3 325.0 352.7

+48.2 +3.5 39.6

ns ns ns ns

ns ns ns

ns: no signi¢cant di¡erence. E2 , 17-L-estradiol; PG, progesterone. a Versus sham-operated group. b Versus ovariectomized (OVX) group.

In contrast to the uterus, ovariectomy did not decrease NGF protein content within the submandibular salivary gland, but treatment of ovariectomized mice with estrogen or progesterone alone or in combination stimulated a signi¢cant increase in NGF protein compared to concentrations in ovariectomized mice, and treatment of ovariectomized mice with estrogen alone or in conjunction with progesterone caused a signi¢cant increase in NGF relative to that observed in intact sham-operated controls (Table 1). Ovariectomy alone or followed by estrogen treatment did not result in NGF mRNA concentrations in the submandibular salivary glands that were signi¢cantly di¡erent from those observed in intact

sham-operated controls, but treatment of ovariectomized mice with progesterone alone or in combination with estrogen stimulated NGF mRNA concentrations in the submandibular salivary glands that were signi¢cantly greater than those observed in intact sham-operated controls, ovariectomized, or ovariectomized/estrogen-treated (Table 2). Similar to the salivary gland, there was no di¡erence between NGF protein content in hearts from intact sham-operated controls and ovariectomized mice, but treatment of ovariectomized mice with estrogen alone or in combination with progesterone stimulated a signi¢cant increase in NGF protein content of the heart com-

Table 2. NGF mRNA content in tissue (NGF/L19 ratio)

Uterus

Salivary gland

Heart

Bladder

Treatment

n

Mean þ S.E.M.

sham OVX OVX+E2 OVX+PG OVX+E2+PG sham OVX OVX+E2 OVX+PG OVX+E2+PG sham OVX OVX+E2 OVX+PG OVX+E2+PG sham OVX OVX+E2 OVX+PG OVX+E2+PG

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 5 6

0.24 þ 0.01 0.15 þ 0.001 0.29 þ 0.001 0.44 þ 0.002 0.87 þ 0.03 0.61 þ 0.10 0.77 þ 0.06 0.95 þ 0.04 1.73 þ 0.40 1.53 þ 0.12 1.39 þ 0.07 1.42 þ 0.04 1.37 þ 0.10 1.62 þ 0.04 1.33 þ 0.03 1.17 þ 0.04 1.37 þ 0.06 1.51 þ 0.08 1.51 þ 0.03 1.32 þ 0.03

Change (%)a

Change (%)b

P*

P**

337.5 +20.8 +83.3 +262.5

+93.3 +193.3 +480.0

6 0.001 6 0.001 6 0.001 6 0.001

6 0.001 6 0.001 6 0.001

+26.2 +55.7 +183.6 +150.8

+23.4 +124.7 +98.7

ns 6 0.001 6 0.001 6 0.001

ns 6 0.001 6 0.001

+2.2 31.4 +16.5 34.3

33.5 +14.8 36.3

ns ns ns ns

ns ns ns

+17.1 +29.1 +29.1 +12.8

+10.2 +10.2 33.6

ns ns ns ns

ns ns ns

ns: no signi¢cant di¡erence. E2 , 17-L-estradiol ; PG, progesterone. a Versus sham-operated group. b Versus ovariectomized (OVX) group.

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pared to concentrations in intact sham-operated controls or ovariectomized mice (Table 1). There were no di¡erences in NGF mRNA abundance among intact sham-operated controls and ovariectomized mice with or without hormone treatment (Table 2). Ovariectomy alone or in combination with hormone treatment did not a¡ect NGF protein content or NGF mRNA in the bladder (Tables 1 and 2).

DISCUSSION

These results indicate that under certain conditions, estrogen and progesterone can increase abundance of NGF mRNA and protein in peripheral organs, and further that this response varies among organs. In general, there was a weak correlation between NGF protein and NGF mRNA. This may be related to the timing of sample collection, because it has been observed that administration of estrogen to ovariectomized mice resulted in an increase in mRNA for heparin-binding epidermal growth factor in the uterus that peaked 2 h after injection and regressed to baseline concentrations by 12 h (Wang et al., 1994). The relative abundance of mRNA does not always correlate with concentrations of the corresponding protein, and di¡erences have been attributed to: (1) stability of mRNA transcripts; (2) relative e¤ciency of translation; (3) post-translational process (most likely protein^protein interaction) and (4) £uctuations in protein stability or degradation (Vizzard et al., 2000). The results of the current study should be considered to re£ect the experimental conditions used, and it is highly probable that the use of di¡erent doses of hormones or sampling times would produce di¡erent results. Establishment of a dose^ response relationship or the time course of responses was not within the scope of the current work. The e¡ects of steroidal hormones on NGF mRNA in the uterus and submandibular salivary gland are particularly intriguing, because they suggest that progesterone alone or in combination with estrogen has the capacity to increase translation of the NGF gene or stability of NGF protein. The concept that ovarian hormones may regulate expression of NGF in peripheral organs is consistent with previous reports indicating that concentrations of NGF message and protein in the uterus £uctuate during pregnancy (Brauer et al., 2000; Varol et al., 2000). NGF protein (normalized to organ weight) and mRNA (normalized to total RNA) decreased signi¢cantly during mid (day 14) and late (day 21) term pregnancy in rats, and relative uterine NGF protein and mRNA concentrations returned to normal by 7 days post-partum (Varol et al., 2000). A similar study performed in guinea-pigs found that uterine NGF protein (normalized to organ weight and protein content) did not £uctuate during pregnancy but did increase post-partum (Brauer et al., 2000) Although these reports provide con£icting data regarding whether or not a decrease in NGF protein may contribute to loss of sympathetic innervation during pregnancy, the combined observations strongly suggest that post-partum synthesis of NGF by

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the uterus may contribute to restoration of sympathetic innervation. It has been observed that NGF protein synthesis may vary among tissue sub-types within the uterus during pregnancy (Varol et al., 2000), and that the content of NGF within the uterus varies among the tubal ends of the horns, the mid-region of the horns, and the cervix (Brauer et al., 2000; Varol et al., 2000). These di¡erences were most evident when NGF content was normalized to organ weight, and it was observed that total uterine NGF (i.e. combined from all portions of the uterus) was relatively constant in immature and mature rats (Varol et al., 2000). The variation in relative abundance of NGF and response in the organs studied may re£ect £uctuations in tissue weight (particularly £uid content), the relative density and activity of ER and PR, and the capacity of speci¢c cell types to respond to ligand activation of these receptors. All the organs we studied express ER and PR. Two primary sub-types of estrogen receptors have been identi¢ed, ERK and ERL, and both sub-types have been detected in a variety of tissues, including the uterus, bladder, salivary glands, and the heart (Camacho-Arroyo et al., 1999; Grohe et al., 1997; Kuiper et al., 1996; Mosselman et al., 1996; Tremblay et al., 1997). It has been observed that ER sub-types are expressed in di¡erent cell types within a single organ and that the relative density of ER isoforms may vary during the estrus cycle, strongly suggesting that ERK and ERL may have di¡erent biological functions (Kuiper et al., 1998; Pelletier, 2000; Wang et al., 2000). The distribution of ER and PR within the bladder also varies with the anatomical region (i.e. trigone versus dome) (Bussolati et al., 1990; Pacchioni et al., 1992; Wolf et al., 1991). The e¡ects of selective estrogen receptor modulators are highly tissue-speci¢c and appear to depend upon the relative abundance and activity of ER sub-types and the diversity of ER target genes (Burger, 2000; Dutertre and Smith, 2000). This may be the basis for the remarkably di¡erent e¡ects of ER antagonists on various organs. PR are expressed constitutively in the stroma and smooth muscle of the uterus, smooth muscle cells of the urinary bladder, and the epithelium of the submandibular salivary gland (Camacho-Arroyo et al., 1999; Uotinen et al., 1999). We also detected abundant staining of PR in the bladder epithelium. The abundance of PR in the uterus £uctuates during the menstrual cycle and pregnancy, apparently in response to estrogen (Meikle et al., 2000; Salamonsen et al., 1999). In bladder biopsies obtained from women, PRs were present in the sub-epithelial region, but their density was highly variable, and signi¢cantly fewer PR were observed in bladder biopsies obtained from post-menopausal women who were not receiving estrogen replacement (Blakeman et al., 2000). PR have been identi¢ed in the myocardium, atrial appendages, and coronary vessels, and estrogen also appears to increase abundance of PR in these structures (Grohe et al., 1997; Ingegno et al., 1988). Although we did not attempt to quantify abundance of ER and PR in this study, the patterns of staining generally re£ected those described previously. Staining

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for PR in the uterus and salivary gland occurred predominantly in the stroma and ducts, respectively; however, PR staining was also observed in epithelium and secretory cells, respectively, in conjunction with staining for NGF, ERK, and ERL in the uterus and salivary gland. It is also possible that progesterone exerted e¡ects on NGF via glucocorticoid receptors. Glucocorticoids stimulate increased expression of NGF, trkA, and p75 in the brain and spinal cord (De Nicola et al., 1998; Grundy et al., 2001; Shi and Mocchetti, 2000), and progesterone binds to the glucocorticoid receptors, albeit with a lower a¤nity than glucocorticoids (Rupprecht et al., 1993). Progesterone or glucocorticoids alone were found to inhibit proliferation of uterine epithelial cells, and although this e¡ect appeared to be mediated by interaction of each compound with its speci¢c cognate receptor, the possibility of interaction of progesterone with glucocorticoid receptors could not be eliminated (Bigsby and Young, 1993). Although PR and glucocorticoid receptors bind to the same DNA sequences (hormone response elements) to induce gene transcription (Lieberman et al., 1993; Roche et al., 1992), the net e¡ect of hormone response element binding is speci¢c to PR or glucocorticoid receptors. Subtle di¡erences in hormone response element sequence or a variety of cofactors (repressors or activators) may account for the speci¢city of response to ligand binding to PR or glucocorticoid receptors (reviewed in Song et al., 2001). Regardless, the possibility remains that progesterone may exert e¡ects via interaction with glucocorticoid receptors. Estrogen has also been demonstrated to have transcriptional e¡ects independent of classical estrogen receptor binding (Watters et al., 1997; Watters and Dorsa, 1998). Estrogen was shown to stimulate expression of neuronal neurotensin mRNA by preoptic neurons via a cAMP/protein kinase A-dependent pathway independent of activation of a recognized estrogen response element (Watters and Dorsa, 1998). It has also been proposed that binding of estrogen to ER at the plasma membrane may result in rapid activation of phosphoinositol 3-kinase and protein kinase B/AKT and subsequent stimulation of extracellular-regulated kinase and p38 mitogen-activated protein kinase (Kelly and Levin, 2001). Modulation of NGF expression by ligand binding to plasma membrane-associated hormonal receptors has not been described but remains a possibility. The e¡ects of NGF on the central and peripheral nervous systems are mediated by interaction with two transmembrane glycoprotein receptors, trkA and p75 (Barbacid, 1994; Chao, 1994). TrkA has high a¤nity and speci¢city for NGF, while p75 binds to a variety of neurotrophins with lower a¤nity. Binding of NGF to trkA triggers a series of protein phosphorylations thought to be essential for signal transduction of NGF (Kaplan and Stephens, 1994). It has been suggested that the e¡ects of ligand binding to p75 are limited to enhanced tyrosine autophosphorylation of trkA subsequent to binding NGF (Verdi et al., 1994), but p75 may be a cellular death signaling protein, because inhibition of p75 expression with anti-sense oligonucleotides

prevented death of neurons in severed sensory nerves (Cheema et al., 1996). Migration of Schwann cells (that do not express the trkA receptor) in developing or regenerating peripheral nerves is modulated by NGF via binding with the p75 receptor (Anton et al., 1994). In addition to their presence in central and peripheral nerves, p75 is relatively ubiquitous in peripheral organs, and trkA has been identi¢ed in a wide variety of tissues, including the uterus, salivary gland, heart, kidneys, spleen, thymus, cornea, gastric parietal cells, the putative intestinal neuroendocrine cells, pancreas (sub-intralobular ducts), adrenal cortex, prostate, the mammary ducts, skin, thymus, lymph node, muscle, and lung (Guate et al., 1999; Lockhart et al., 1997; Lomen-Hoerth and Shooter, 1995; Shibayama and Koizumi, 1996). The widespread expression of neurotrophin receptors in areas not known to be targets for neurotrophins supports the hypothesis that neurotrophins may have broader functions outside the nervous system (LomenHoerth and Shooter, 1995). It is further interesting to note that short-term estrogen replacement (two injections at 24-h intervals 24 h prior to being killed) in ovariectomized rats signi¢cantly increased trkA mRNA in the lumbar dorsal root ganglia. The e¡ect was not dependent upon target organ-derived NGF, because ipsilateral sciatic axotomy did not prevent increased trkA in response to systemic estrogen (Liuzzi et al., 1999). This suggests that steroidal hormones may also modulate the response of peripheral tissues to NGF by regulating abundance of its high a¤nity receptor. The exact signi¢cance of regulation of NGF in peripheral organs by steroidal hormones remains unclear. Cellular degeneration and repair within the uterus during the menstrual cycle has been compare to wound healing (Salamonsen et al., 1999), and steroidal hormones stimulate synthesis and release of many growth factors within the uterus, including epidermal growth factor, vascular endothelial growth factor, transforming growth factor-K, and insulin-like growth factor-1 (Ace and Okulicz, 1995; Greb et al., 1997; Hyder et al., 2000; Klotz et al., 2000; Niikura et al., 1996). Treatment of immature female mice with NGF stimulated an increase in uterine weight and enhanced the hypertrophic response of the uterus to chorionic gonadotropin (Akasu et al., 1970).

CONCLUSION

The results of our study indicate that under certain conditions, estrogen and progesterone have the capacity to up-regulate NGF mRNA and protein in peripheral organs, speci¢cally the uterus and submandibular salivary gland. The results of the current study must be viewed in light of the fact that the experiments were conducted using a single dosing regimen and a single end point after hormone administration. Di¡erential responses among organs suggests that either a threshold number of estrogen and/or PRs are required to initiate this response or that cofactors present within certain cells augment the e¡ects of steroidal hormones on NGF synthesis. Other organs of the urogenital tract were not

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evaluated, but it is possible that they may respond in a similar manner. Increased abundance of NGF in peripheral organs in response to steroidal hormones may mod-

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ulate the response to painful or in£ammatory stimuli and, if prolonged, could contribute to neoinnervation of target organs.

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