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Endocrinology Vol. 140, No. 7 3003-3011
Copyright © 1999 by The Endocrine Society

ARTICLES

Thyroid Hormone (3,5,3'-Triido-L-Thyronine) Masking/Inversion of Stimulatory Effect of Androgen on Expression of mk1, a True Tissue Kallikrein, in the Mouse Submandibular Gland1

Kinji Kurihara, Shichiro Maruyama, Nobuo Nakanishi, Hiroshi Sakagami and Takao Ueha

Departments of Oral Physiology (K.K., T.U.), Dental Pharmacology (S.M., H.S.) and Biochemistry (N.N.), Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan

Address all correspondence and requests for reprints to: Kinji Kurihara Ph.D., Department of Oral Physiology, Meikai University School of Dentistry, 1–1 Keyaki-Dai, Sakado, Saitama 350-0283 Japan. E-mail: kkinji{at}dent.meikai.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We studied hormonal regulation of the expression of mk1, a true tissue kallikrein, in the submandibular gland (SMG) of ICR, C3H/HeN, and F1 (mice from male C3H/HeN x female ICR and in the ones from male ICR x female C3H/HeN). In these mouse strains, mk1 was low in content in males, abundant in females, and increased remarkably by castration of males. In the case of ICR and both F1 mice, injection of 5{alpha}-dihydrotestosterone (DHT) reduced the mk1 level of castrated and female mice. However, the mk1 content in female C3H/HeN mice (or castrated C3H/HeN) was further increased by DHT. To investigate the real action of DHT on mk1 expression, we examined the effects of adrenoectomy/glucocorticoid (dexamethasone, Dex) administration; DHT administration into castrated and adrenoectomized mice; ovariectomy/female hormone (17ß-estradiol, progesterone) administration; and hypophysectomy/combinatory administra-tion of DHT, Dex, and thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) on the mk1 expression in the SMG of ICR mice. Adrenoectomy or ovariectomy did not change the characteristic pattern of mk1 expression in male and female ICR mice. In hypophysectomized (Hypox) ICR male mice, the mk1 content was increased to the same level as in normal ICR females, and DHT administration into the Hypox mice further increased the mk1 level. However, combinatory administration of DHT + T3 or of DHT + T3 + Dex into the Hypox mice lowered the mk1 content to the level of normal ICR males, whereas T3 single administration had no effect. Dex single administration into the Hypox mice increased the mk1 level to an even higher than that observed with DHT administration. The mk1 level in Hypox mice was not significantly changed by coadministration of Dex with T3. From these results, we conclude that 1) mk1 expression is fundamentally stimulated by androgen (DHT) as are other mk isozymes, such as mk9, mk13, mk22, and mk26 in the mouse SMG, 2) the effect (stimulatory) of DHT on mk1 expression becomes, however, inverted (inhibitory) in the presence of T3. Although the serum T3 level of C3H/HeN female (0.52 ng/ml) was not significantly different from that of C3H/HeN males or ICR mice, coadministration of T3 into C3H/HeN females with a fixed amount of DHT (20 mg/kg body weight) dose dependently repressed the DHT-induced increase in mk1 expression, suggesting the lower sensitivity of C3H/HeN females to T3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL known that the SMG of the male mouse contains many important bioactive substances such as nerve growth factor (NGF) (1), epidermal growth factor (EGF) (2), and renin (3). In addition to these three bioactive substances, the mouse SMG has various esteroproteinase isozymes, members of mouse kallikrein gene family: mk1 (proteinase F, pI 4.7), mk9 (proteinase D, pI 5.7), mk22 (proteinase A, pI 5.9), mk13 (P-esterase, pI 9.9) (4, 5, 6, 7), and mk26 (pI 6.6 protein) (Kurihara, K., unpublished data). Mk9 (8), mk22 (9, 10, 11), mk13 (12, 13, 14), and mk26 (15) were found to be identical to EGF binding protein, ß-NGF endopeptidase, and prorenin-converting enzyme 1 and 2, respectively. Mk1 is thought to be a true tissue kallikrein because mk1 has the strongest kininogenase activities for both low molecular weight and high molecular weight kininogens, whereas mk22 has 1/6 and 1/50 the activity of mk1 for the respective kininogens as its substrate (7). Kininogenase activities of mk9 and mk13 are less than 1/100 of the activity of mk1 for both substrates (7). Thus, mk1 is the most important one possessing kallikrein function in the kallikrein gene super family.

The SMG of mice is known to have androgen receptors (16, 17) and to show eminent sexual differences (3, 18, 19). The most prominent characteristic of NGF, EGF, renin and mouse kallikrein (mk) isozymes in the mouse SMG is a sexual difference in their content. Expression of mk9, mk13, mk22 (20, 21, 22, 23, 24, 25), and mk26 (Kurihara, K., unpublished results) is androgen inducible. The effect of androgen on the expression of these bioactive substances (NGF, EGF, renin, and mk isozymes) is modulated by other hormones such as glucocorticoid (26, 27) and thyroid hormone (28, 29). Thus, the mouse SMG provides us an interesting and useful model system for studies on regulatory mechanisms of gene expression of bioactive substances by androgen and other hormones.

On the other hand, mk1 is a unique and exceptional isozyme among the members of the mk gene family; e.g. in the ICR mouse strain, the mk1 content is much higher in the female SMG than in the male one; castration increases its content to the level of normal females; and 5{alpha}-dihydrotestosterone (DHT)-administration to females or castrated males causes a decrease in mk1 content (30, 31). Thus, DHT was thought to have an inhibitory effect on the mk1 expression. However, in the present study, we found in C3H/HeN mouse that DHT administration to females or castrated males further increased the mk1 expression in the SMG. The results suggest a possibility that androgen has originally stimulatory action on the expression of mk1 and that this stimulatory effect of androgen is inverted to the opposite direction in the copresence of other hormone(s). It seems likely that mk1 expression is fundamentally stimulated by androgen because the other members of mk gene super family, mk9, mk13, mk22 (20, 21, 22, 23, 24, 25) and mk26 (Kurihara, K., unpublished data) in the mouse SMG are all androgen inducible. Furthermore, the effect of DHT on expression of these androgen-inducible mk isozymes is reported to be affected by glucocorticoid, thyroid, and female hormones (26, 27, 28, 29, 32). Thus, the above finding suggests a novel and unique mechanism underlying the regulation of mk1 expression in the mouse SMG, in which the stimulatory effect of androgen is inverted to become inhibitory by some other hormone(s).

We examined the combinatory effect of various hormones on mk1 expression by using model animals in which levels of various hormones were diminished by castration (Cast), ovariectomy (Ovx), adrenoectomy (Adex), and hypophysectomy (Hypox). We found that DHT had a stimulatory effect on mk1 expression when the pituitary-dependent hormones are diminished by Hypox. Furthermore, coadministration of T3 with DHT decreased the mk1 level of Hypox mice to one lower than that of control animals (Hypox mice without any administration); though T3 single administration had neither a stimulatory nor inhibitory effect. Thus, T3 was found to be the hormone that inverts the fundamental action of androgen (stimulatory effect) on mk1 expression in the mouse SMG to the opposite direction (inhibitory).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Ampholine carrier ampholytes were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Benzoylarginine ethylester (BAEE), alcoholdehydrogenase, and NAD were purchased from Sigma Chem-ical Co. (St. Louis, MO). N-succinimidyl3-(4-hydroxy-5-[125I]iodophenyl)propionate (Bolton and Hunter reagent, 500 µCi) and immobilized antirabbit IgG donkey IgG (Amerlex-M) were from Amersham Pharmacia Biotech (Buckinghamshire, UK). Other reagents were obtained from Wako Pure Chemicals (Osaka, Japan).

Animals and treatments
Male and female mice from ICR and C3H/HeN strain were purchased from Japan Clea (Tokyo, Japan). All experiments were carried out in accordance with the guidelines for animal experiments of Kyoto University (1988). All animals were killed at 10 weeks of age in all of the experiments. Two types of F1 mice were prepared by mating as follows: F1(female ICR x male C3H/HeN) and F1(female C3H/HeN x male ICR).

Cast, Ovx, and Adex were carried out under ether anesthesia 4 weeks before hormone treatments.

Male ICR mice of 6 weeks of age were sent to Teikoku-Hormone Manufacturing Co., Ltd. (Kanagawa, Japan) for hypophysectomy via the auditory canal under pentobarbital anesthesia. The Hypox mice were returned to our facilities and maintained under standard laboratory conditions for 28 days before hormone treatments were begun.

DHT (6 mg/ml), dexamethasone (Dex, 3 mg/ml), 17ß -estradiol (E2, 0.33 mg/ml), and progesterone (Pro, 3 mg/ml) were suspended in sesame oil; and T3, i.e. 3,5,3'-triiodo-L-thyronine (T3, 0.3 mg/ml), was suspended in 0.9% NaCl and solubilized by addition of NaOH to a final concentration of 0.005 N. All hormones were injected sc 5 times with a 1-day interval between injections at the following dose: DHT, 20 mg/kg; Dex, 10 mg/kg; E2, 1 mg/kg; Pro, 10 mg/kg; T3, 1 mg/kg. On the day after the last hormone injection, the animals were killed by cervical dislocation; and their SMGs were removed. Each SMG was homogenized with a Teflon pestle glass homogenizer (Potter Elvehjem type) containing 20 mM sodium phosphate buffer (pH 7.0). The homogenate was centrifuged at 29,700 x g for 30 min, and the resulting supernatant was used for assays.

Isoelectric fractionation
Isoelectric fractionation of the crude extract of the SMG was carried out by the method of Vesterberg and Svensson (33). A sucrose gradient from 0 to 50% Ampholine carrier ampholytes was prepared in a 65-ml column for electric focusing. The SMG extract was loaded into the middle of the gradient and electric focused for 24 h at 2 C at 700 V. Samples after electric focusing were collected as 1-ml fractions.

Measurement of esteroproteinase activity
The esteroproteinase activity was assayed by measuring the increase in absorbency at 340 nm after coupling to the NAD+-alcohol dehydrogenase system, with benzoylarginine ethylester (BAEE) used as a substrate (34).

RIA of mk1
Mk1 (proteinase F) was purified from 10-week-old female ICR mice, and antiserum for mk1 was prepared as described in a previous paper (30).

Five micrograms of mk1 protein in 20 µl of borate buffer (pH 8.5) was labeled with 500 µCi of N-succinimidyl3-(4-hydroxy-3,5-di[125I]iodo-phenyl)propionate (35) by incubation at 4 C overnight. The labeled protein was separated from uncoupled radioisotope by gel filtration using Sephadex G-50 equilibrated with 50 mM sodium phosphate buffer containing 0.2% gelatin (pH 7.4). And then 50 µl of assay buffer (PBS containing 0.5% BSA and 25 mM EDTA), 50 µl of labeled mk1 protein (approximately 30,000 cpm), 50 µl of anti-mk1 antiserum, and 50 µl of sample or purified standard mk1 protein (0.01–500 ng) were combined and incubated overnight at 4 C. Two hundred and fifty microliters of immobilized antirabbit IgG donkey IgG (Amerlex-M) was added, and the whole mixture was incubated at room temperature for 15 min. The mixture was then centrifuged, and the immunoprecipitated radioactivity was measured in an Aloka Auto Well {gamma} System, ARI-500 (Tokyo, Japan).

Measurement of serum T3
Blood was collected from the inferior vena cava under ether anesthesia, and the serum was separated by centrifugation. The serum T3 concentration was measured by SRL Tokyo Medical Co., Ltd. (Tokyo, Japan) by use of a solid-phase [125I]RIA, T-3 RIABEAD T3 Assay Kit produced by DAINABOT Co., Ltd. (Tokyo, Japan).

Measurement of protein
Protein contents were measured with a Bio-Rad Protein Assay Kit produced by Bio-Rad Laboratories, Inc. (Richmond, CA), with BSA as the standard (36).

Statistical analysis
Statistical analyses were done with a computer package (Microsoft Corp. Excel). Data were presented as means and SE (means ± SE). Levels of statistical significance between means were calculated by the two-tailed Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of androgen on mk1 and other esteroproteinases (mouse tissue kallikrein gene family) in the SMGs of ICR, C3H/HeN, and F1 mice
As shown in Fig. 1AGo, total activity of esteroproteinases in the SMGs of ICR, C3H/HeN, and F1s from these two strains showed the following characteristics: 1) male mice had far larger total esteroproteinase activity than females; 2) castration of males decreased the esteroproteinase activity to the same level as in females; 3) administration of DHT to the castrated mice recovered the esteroproteinase level to that of the normal males; 4) DHT administration to female mice increased the esteroproteinase activity to that of the male level.



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  		Figure 1. A, Effects of Cast and injection of DHT on total esteroproteinase activity in the SMG of ICR, C3H/HeN, and F1(female ICR x male C3H/HeN and female C3H/HeN x male ICR) mice. Each datum indicates the mean ± SE from three to seven mice. *, P < 0.001, significant difference by Student’s t test. B, Effects of castration and injection of DHT on the mk1 content in the SMG of ICR, C3H/HeN, and F1 (female ICR x male C3H/HeN and female C3H/HeN x male ICR) mice. Each datum indicates the mean ± SE from three to seven mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001, significant difference by Student’s t test.

 
There are (mainly) five types of mouse kallikrein isozymes: mk1, mk9, mk13, mk22, and pI 6.6 proteinase. The pI 6.6 enzyme has been identified as mk26 by amino acid analysis (Kurihara, K., unpublished results). Because expression of the majority of the mk isozymes was dependent on androgen and the activity of mk1 was about 2.27%, 22.5%, 3.4%, and 24.4% of the total protease activities in the SMGs of ICR male, ICR female, C3H/HeN male, and C3H/HeN female mouse, respectively (Fig. 2Go), total esteroproteinase activity was far greater in males than in females (Fig. 1AGo). However, the mk1 isozyme appeared to be a unique member of the mouse kallikrein gene family.



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  		Figure 2. Isoelectric focusing patterns of the esteroproteinase activity and mk1 content in the SMG of ICR, C3H/HeN, and F1(female ICR x male C3H/HeN and female C3H/HeN x male ICR) mice. SMG extracts from each strain were subjected to IEF (pH range of 3–10). 100 U of esteroproteinase activity was loaded onto the column. Yields of esteroproteinase activity in all experiments were 90–110% after IEF.

 
Mk1 expression was found to be inhibited by DHT: 1) mk1 was exceptionally more abundant in female mice than in male ones (Fig. 2Go); and 2) castration of males increased the mk1 level to a value even higher than that of females (Fig. 1BGo). However, we found, in C3H/HeN female and castrated C3H/HeN mice, that DHT administration increased the mk1 content (Fig. 1BGo). On the other hand, effects of castration and DHT administration on the other mks (mk9 and mk26) in the C3H/HeN mouse were the same as in the ICR mouse (data not shown). We also analyzed the effect of androgen on mk1 contents and the kallikrein isozyme patterns in F1 mice, to examine which characteristics of the two strains are inherited by their offsprings (Figs. 1Go and 2Go). In terms of isozyme patterns, F1 mice expressed characteristics of both ICR and C3H/HeN: they had a substantial amounts of mk26 (pI 6.6 isozyme), which is abundant in the C3H/HeN strain but not in the ICR strain, and mk13, which is found in ICR mice but not in C3H/HeN ones. With respect to the mk1 expression, however, both F1 mice showed the ICR mouse-type response: female mice of either F1(ICR female x C3H/HeN male) or F1(C3H/HeN female x ICR male) showed the same response to DHT as ICR female in terms of mk1 expression (Fig. 1BGo), and had the same mouse kallikrein isozyme patterns with each other. This was also true for both F1 males (Fig. 2Go).

Effects of Adex/glucocorticoid and of Ovx/female hormones on mk1 expression
Because androgen (DHT) worked in a stimulatory fashion on mk1 expression in C3H/HeN females (and C3H/HeN castrated males) but in an inhibitory one in the ICR strain, we postulated the following working hypothesis: 1) mk1 expression is originally (fundamentally) stimulated by androgen (DHT), and 2) this effect of DHT is modulated and turned in the opposite direction in ICR mice by some factor(s), probably hormones from the adrenal cortex, thyroid gland, and/or ovary.

To test our hypothesis, we examined the effect of Adex and glucocorticoid administration on ICR mice (Fig. 3Go). Adex of ICR male mice did not change their mk1 expression, and Dex administration to the Adex ICR males failed to have any significant effect. On the other hand, Adex of ICR females slightly increased their mk1 level, and this increase was reversed by Dex administration. These results suggested a slight inhibitory effect of Dex on mk1 expression in the ICR female mouse, but either removal of adrenal hormones or administration of Dex did not invert the characteristic mk1 expression in males and females.



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  		Figure 3. Effects of Adex and injection of Dex on mk1 content in the SMG of ICR mice. Each datum indicates the mean ± SE from five to seven mice. P < 0.05, P < 0.001, significantly difference by Student’s t test.

 
We also examined the time course of the effect of DHT administration on mk isozyme contents in castrated-adrenalectomized (Cast-Adex) ICR mice (Fig. 4Go). Activity of each mk isozyme was analyzed after separation by isoelectric focusing. At day 3, activities of mk9, mk13, and mk22 started to increase and reached maximum levels on the fifth day. On the other hand, mk1 showed the opposite response to DHT. Thus, the results with Adex and Cast-Adex mice suggest that the effect of androgen on mk1 expression was not significantly modified by endogenous glucocorticoid.



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  		Figure 4. Time-course of the effect of DHT administration on mk1, mk9, mk13, and mk22 activities in the SMG of Cast-Adex ICR mice. DHT was injected sc at the dose of 20 mg/kg body weight. On the day described in this figure, the animals were killed by cervical dislocation. For this experiment, the SMGs from five mice were pooled for each group, and then 20 U of total esteroproteinases was subjected to IEF (pH 4–6 ampholine carrier ampholytes). Data indicate the mean of triplicate experiments. The esteroproteinase activity of each isozyme was obtained by multiplying the percent ratio of each proteinase isozyme separated by IEF by the total esteroproteinase activity.

 
We then examined the effect of Ovx and female hormone administration (Fig. 5Go). Either E2 or Pro failed to have a significant effect on the mk1 level of ICR male mice. Removal of female hormones from ICR females by Ovx did not decrease the mk1 content, in clear contrast to the effect of DHT administration, which lowered the mk1 level of ICR females. E2 administration to Ovx-ICR females had no effect and Pro appeared to slightly increase the mk1 content, but this increase was not statistically significant in this experiment. These results suggested that female hormones might not be a primary factor to determine the level of mk1 expression; i.e. the high level of mk1 in ICR females was not due to the presence of ovaries (female hormones) nor the low level of mk1 in ICR males due to the absence of female hormones.



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  		Figure 5. Effects of Ovx and injection of E2 or Pro on the mk1 content in the SMG of ICR mice. Each datum indicates the mean ± SE from four to eight mice. P < 0.001, significant difference by Student’s t test.

 
Effect of Hypox and inversion of androgen (DHT) action by T3
We examined the effect of various hormones on mk1 expression in Hypox ICR male mice, by which almost all of the hormones secreted from genital organs, adrenal cortex, and thyroid glands are diminished. The success of the Hypox was evident from the significant reduction in weights of the adrenal glands, seminal vesicles, and testes (data not shown). In addition, the weight of the SMG was significantly decreased by about 30% (data not shown), consistent with our previous studies (32, 37). As shown in Fig. 6Go, Hypox of the ICR male mouse increased the mk1 expression to the same level as in normal ICR females. Moreover, upon the administration of DHT, the mk1 content in Hypox ICR males was increased to a much higher level, verifying our assumption that androgen (DHT) has fundamentally a stimulatory effect on mk1 expression in the SMG of either C3H/HeN or ICR strain. The results also suggest that these Hypox ICR males are quite useful as an experimental model to examine our hypothesis in which stimulatory action of androgen (DHT) is inverted by the coexistence of some other hormone(s).



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  		Figure 6. Effects of Hypox and injection of DHT, Dex or T3 on the mk1 content in the SMG of ICR mice. Control is Hypox male mouse. Each datum indicates the mean ± SE from three to four mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001, significant difference by Student’s t test.

 
With this model animal (Hypox ICR male mouse), first we tried to reconstitute normal ICR male from Hypox males by administering the three major hormones, DHT, Dex, and T3, to them. Indeed, the administration of DHT in combination with Dex and T3 decreased the mk1 level to a half of that of normal ICR females (or Hypox ICR males), to approximately one-third of that observed in Hypox ICR males administered DHT, and to a level comparable with that of normal ICR males.

We then examined which hormone(s), Dex or T3 or both of them, inverts the action of DHT on mk1 expression. Dex showed stimulatory effect when administered alone to the Hypox ICR males; in fact, stimulation by Dex was even stronger than that by DHT. Dex did not have an additive effect with DHT when used in combination. Stimulatory effect of Dex seemed to be neutralized in the presence of DHT rather than the stimulatory effect of DHT being inhibited by Dex. These results are compatible with those obtained in the former experiments shown in Figs. 3Go and 4Go. On the contrary, T3 lowered the mk1 content nearly to the level observed in normal ICR male mice when used in combination with DHT, whereas T3 single administration to Hypox ICR males was neither stimulatory nor inhibitory. These results indicate that T3 is the very hormone that inverts the stimulatory effect of androgen (DHT) on the mk1 expression in the SMG to quite the opposite direction, inhibitory. It should be noted that T3 appeared to partially inhibit Dex-induced increase in mk1 content when used in combination with Dex; i.e. mk1 in the [Hypox + Dex + T3] mouse was less than that in the [Hypox + Dex] mouse. However, the mk1 level in [Hypox + Dex + T3] was still the same as that in normal ICR females, Hypox ICR males, and/or [Hypox + T3] mice. In other words, T3 might neutralize the stimulatory effect of Dex but not invert the Dex effect at all. Thus the inverting action of T3 is rather specific for androgen (DHT).

Thyroid hormone levels and effect of T3 administration on mk1 expression in the C3H/HeN female mouse
We examined whether the T3 level of C3H/HeN female mice is less than that of C3H/HeN males and/or ICR mice because the hormonal environment of C3H/HeN females with respect to T3 seemed to be somehow similar to those of Hypox ICR mice: they showed a similar response to DHT for mk1 expression. However, C3H/HeN females had almost the same T3 level as normal C3H/HeN males, ICR males, and ICR females (Table 1Go).


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  		Table 1. Serum T3 levels of ICR and C3H/HeN mice

 
These results suggest a possibility that the C3H/HeN female mouse might have lower sensitivity to T3 so that endogenous T3 is not enough to invert the stimulatory action of DHT on mk1 expression. Therefore, we examined the effect of T3 administration on the androgen (or DHT)-dependent increase in mk1 expression (Fig. 1BGo) in C3H/HeN female mice. As shown in Fig. 7Go, coadministration of T3 with a fixed amount of DHT dose dependently abolished the stimulatory effect of DHT on mk1 expression in C3H/HeN females, supporting the above assumption that C3H/HeN mice (at least females of this strain) may be less sensitive to T3. EC50 of T3 for inhibition of DHT-dependent increase in mk1 expression was estimated to be about 0.3 mg/kg body weight (Fig. 7Go).



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  		Figure 7. Effects of injection of DHT and DHT + T3 on the mk1 expression in the SMG of C3H/HeN female mice. •, Hormones were injected sc 5 times with a one-day interval between injections at the following dose: DHT, 20 mg/kg body weight combined with T3, 0, 0.001, 0.01, 0.1, 0.5, and 1 mg/kg body weight. {circ}, Normal female mice. For this experiment, the SMGs of five mice of each group were pooled, and then 2 mg protein of SMG supernatant of each group was subjected to IEF (pH 4–6 ampholine carrier ampholytes). Mk1 (pI 4.7) activity was measured after separation by IEF.

 
On the other hand, DHT administration (20 mg/kg body weight) to C3H/HeN male mice did not change the mk1 activity in the SMG (the difference in the mk1 activity between normal males and DHT-administered males was less than 3%).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The mouse submandibular gland (SMG) is abundant in various esteroproteinases (mouse kallikrein isozymes; mk isozymes). The most prominent characteristic of mk isozymes in mouse SMG is a sexual difference in their content. Mk9, mk13, mk22, (20, 21, 22, 23, 24, 25), and mk26 (Kurihara, K., unpublished result) are abundant in males and androgen dependent. On the contrary, mk1 content is greater in the SMG of ICR female mice than in that of males, is increased remarkably by castration, and is decreased by DHT administration to ICR females (30, 31). Thus, the mk1 expression was thought to be inhibited by androgen. However, we found that the C3H/HeN female mouse showed a different response to androgen in terms of mk1 expression: i.e. by the DHT administration, the mk1 content in C3H/HeN females was increased whereas that in ICR females was decreased.

Therefore, to clarify the real action of DHT on mk1 expression, we postulated the working hypothesis that mk1 expression is originally (fundamentally) stimulated by androgen (DHT), and this effect of DHT is modulated and turned in the opposite direction by some factor(s), probably hormones from the adrenal cortex, thyroid gland, and/or genital organs. We examined our hypothesis with ICR mice whose hormones were diminished by Cast, Ovx, Adex, or Hypox. Adex of the ICR male mouse did not increase the mk1 level (Fig. 3Go), indicating that androgen acted as an inhibitor of mk1 expression in ICR males even in the absence of glucocorticoid. Therefore, glucocorticoid was not a hormone that inverted the stimulatory action of androgen on mk1 expression to make it inhibitory. This was further supported by the results shown in Fig. 4Go, where the effect of DHT administration to Cast-Adex ICR mice was examined. The mk1 content in the SMG of these mice was decreased by DHT administration, indicating that DHT acts in an inhibitory manner on mk1 expression in the absence of endogenous glucocorticoid. On the other hand, administration of female hormone (E2 or Pro) to ICR males did not increase the mk1 level, and Ovx of ICR females did not decrease their mk1 content (Fig. 5Go), indicating that female hormones are not the primary factor that determines the mk1 expression in the presence and even in the absence of endogenous androgen.

To examine the effect of thyroid hormone, T3, we employed Hypox because the removal of the thyroid gland was rather technically difficult. Furthermore, Hypox has the advantage of completely depleting pituitary gland-dependent hormones such as sex hormones, glucocorticoid, and thyroid hormones at the same time (Fig. 6Go). By the Hypox of ICR male mice, the mk1 content increased to the same level as in normal ICR females. The result is compatible with our idea that androgen acts as an inhibitor of mk1 expression under the hormonal conditions (in the presence of other hormones) of normal ICR males. That DHT administration into Hypox ICR males further increased the mk1 level over that observed in control animals (Hypox ICR males without any administration) just coincided with the theoretical consequence deduced from our hypothesis that androgen fundamentally acts as a stimulator of mk1 expression in the absence of other hormones. Coadministration of DHT with T3 into the Hypox mice decreased the mk1 level far lower than that of the Hypox ICR males without any administration and lower than that of normal ICR females, further supporting our hypothesis. Moreover, the result also indicates that T3 is the hormone that lowers the mk1 level of mouse SMG in the presence of DHT by inverting the stimulatory effect of DHT on mk1 expression to become inhibitory, though T3 itself has no inhibitory effect on the mk1 expression in the absence of DHT. Dex administration to the Hypox ICR males also increased their mk1 level. There is a possibility that some part of this Dex effect is, in the absence of androgen, mediated via androgen receptors, since glucocorticoid is reported to bind to androgen receptors in the SMG in a competitive manner with respect to androgen (26, 27). However, coadministration of T3 with Dex to the Hypox mice neutralized the Dex-dependent increase in mk1 content but did not decrease the mk1 level to a value lower than the control one (mk1 in Hypox mice without hormone administration). T3 did not invert the stimulatory effect of Dex. The part of the Dex effect mediated via androgen receptors might be minor, if any. Thus, we have verified our hypothesis that 1) androgen (DHT) has originally a stimulatory action on mk1 expression, and 2) this effect of DHT is turned in the opposite direction by the coexistence of other hormone(s). T3 was identified as the hormone that inverts the DHT action.

Then we examined the reason why C3H/HeN females and ICR females showed a different response to DHT for mk1 expression. Because the response of Hypox ICR males to DHT for mk1 expression was similar to that of C3H/HeN females to the hormone, a lower level of T3 was expected for C3H/HeN females. However, they had the same T3 level as ICR mice and C3H/HeN males (Table 1Go). These values for T3 levels of C3H/HeN and ICR strains are similar to those reported for T3 levels of C3H/HeJ (0.62 ng/ml) and C57BL/6J (0.72 ng/ml) by Maia et al. (38) and for T3 level of ICR (0.72 ng/ml) by Burgi et al. (39). Maia et al. (38) demonstrated that although the C3H/HeJ mouse had less T3 synthesis from T4 because of having less deiodinase activity than the C57BL/6J mouse, C3H/HeJ had almost the same serum T3 level as C57BL/J because of their lesser activity for T3 clearance. The results suggested that the SMG of C3H/HeN mice (at least females) may be less sensitive to T3 for some reason, for example, due to the smaller number of T3 receptors (or some specific T3 receptor subtype) and/or the lower affinity of the receptors to T3. That administration of exogenous T3 with a fixed amount of DHT (20 mg/kg body weight) to C3H/HeN females dose dependently blocked the DHT-induced increase in mk1 expression (Fig. 7Go) supports this assumption. Alternatively there is a possibility that supraphysiological concentrations of DHT caused by DHT administration to females or castrated males of C3H/HeN overcomes the effect of T3, since C3H/HeN females and males have the same serum T3 level. Plasma DHT levels of C3H/HeN males and females were reported to be approximately 2.2 ng/ml and 0.22 ng/ml, respectively, by Angele et al. (40). However, this seems to be unlikely because DHT administration to C3H/HeN males failed to change the mk1 level in their SMG and because DHT administration to ICR females, which also have the same level of serum T3 as C3H/HeN mice (Table 1Go), decreased the mk1 expression (Fig. 1BGo). Thus, the difference in the DHT response for mk1 expression between C3H/HeN females and ICR females may be caused by the lesser sensitivity of C3H/HeN females to T3.

Finally, the question as to why mk1 expression in the C3H/HeN male mouse is less than that in the C3H/HeN female mouse is to be asked. It is important to note that the above issue actually involves two questions that are related to each other. One is why the male mouse (generally) has such a lower mk1 level than the female mouse (this is not a specific phenomenon only for C3H/HeN strain but is also observed in the ICR strain in which female mk1 expression is repressed by DHT administration). And the other is why mk1 in C3H/HeN males who have endogenous androgen (DHT) is less than that in C3H/HeN females whose mk1 expression is further increased by DHT administration. When total activities of esteroproteinases (mks) (Fig. 1AGo) or each mk isozyme other than mk1 (not shown) were compared between normal males (in ICR, C3H/HeN, and F1s) and experimental male models ([Cast + DHT] or [Female + DHT]) of the respective strains, the mk isozyme levels (other than mk1) of male models were similar to those of each normal male, indicating that the expression of mk isozymes (other than mk1) is thought to be primarily and simply determined by DHT. However, in the case of mk1 (Fig. 1BGo), its expression in male models ([Cast + DHT] and [female + DHT] of both ICR and C3H/HeN strain) were considerably (and significantly) different from that in normal males of the respective strains, suggesting a possibility that some unknown testis-derived substance(s) in addition to the combinatory action of DHT and T3 is also involved in the regulation of mk1 expression in the SMG of the normal male mouse. C3H/HeN males and ICR males may similarly respond to such testis-derived substance(s), resulting in a similar low level of mk1 expression. Such a testis-derived substance(s) remains to be elucidated.

On the other hand, it is possible that removal of GH by Hypox somehow affects the physiology of the SMG including production of mk isozymes; and this point should be examined. However, T3 inversion of stimulatory effect of DHT on mk1 expression is observed either in the presence (in the experimental system with C3H/HeN females; Fig. 7Go) or absence of GH (in the experimental system with Hypox mice; Fig. 6Go).

We also examined, in the present study, the mk isozyme patterns and the androgen-response for mk1 expression in SMG of F1 mice (offsprings of female ICR x male C3H/HeN and of female C3H/HeN x male ICR) (Fig. 2Go). Either type of F1 mice had all of the mk isozymes contained in either of the parent strains, whereas both F1s showed the ICR-type response to androgen for mk1 expression. The ICR mouse is known as a closed colony type of CD-1 mice, and C3H/HeN is inbred. Characteristics of ICR in terms of the androgen-response for mk1 expression are presumably dominant over those of C3H/HeN.

The combinatory effect of DHT with T3 demonstrated in the present study might be due to a characteristic genomic structure(s) in the regulatory region of the mk1 gene. Advances in molecular biology have made it possible to clone and analyze the genomic DNAs of many enzymes and proteins with biological activity, and complementary DNAs (cDNAs) of mk1 and of other mk isozymes have been already cloned (13, 15, 41, 42, 43, 44, 45). Therefore, once the characteristic structure of the mk1 gene regulatory element(s) responsible for this novel and unique combinatory action of DHT with T3 is identified, it may help to predict the effect of DHT/T3 on the expression of any other genes that have identical or homologous sequences in their regulatory regions.

As described in the present work, complex phenomena may occur in gene expression when enzymes are regulated by the actions of multiple hormones. Orlowski and Lingrel (46) reported complex interactions of thyroid and glucocorticoid hormones for the expression of Na+,K+-ATPase {alpha} and ß subunits of neonatal rat cardiac myocytes in culture. We also observed the paradoxical effect of T3 and Dex on the Na+,K+-ATPase activity in the SMG of the Hypox mouse: Na+,K+-ATPase activity was increased by single administration of either hormone but was decreased by the combined administration (37). It has been revealed that many genes have multiple elements in their 5'-regulatory regions or in their enhancer regions for binding of gene regulatory proteins, suggesting that expression of many enzymes is regulated by the combined action of multiple factors including growth factors and hormones. The Hypox mouse, in which all pituitary gland-dependent hormones are depleted, provides us a useful model system for analyzing a single hormone action on certain gene expression, which is regulated in normal animals by combinatory actions of multiple hormones.


   Acknowledgments
 
We thank Dr. Edward W. Gresik for helpful discussions and encouragement during the course of this work. We would like to gratefully and respectfully acknowledge the late Dr. Toyoaki Murai for support of our experiments. Also, we thank Dr. Hiroo Takahashi at Teikoku Hormone Mfg. Co., Ltd. for his help with the hypophysectomy of the mice.


   Footnotes
 
1 This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan (Nos. 07672027, 10671748, and 11671851) and by a grant from the Miyata Foundation, Meikai University. Back

Received October 13, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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 J. Histochem. Cytochem.Home page
S. Kurabuchi, E. W. Gresik, and K. Hosoi
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