Hormonal effects on hamster lacrimal gland female-specific major 20 kDa secretory protein and its immunological similarity with submandibular gland major male-specific proteins

Hormonal regulation of a major 20 kDa protein of hamster exorbital lacrimal gland (LG) was studied by SDS-PAGE profile analysis and the purified protein's antisera was used to screen tissues of hamster and other species for crossreacting proteins. This protein was seen in female LG but not in males and late-pregnant or hCG-treated females. Low estrogen state in females after gonadectomy, prolonged light-deprivation, prolonged starvation or lactation increased its level several folds to 20% of LG soluble proteins and similar levels were induced in males after gonadectomy (low androgen state). However, light-deprivation or melatonin treatment-induced low androgen state in males had no effect. In gonadectomized hamsters, this LG protein was obliterated on treatment with androgens, estrogens or thyroid hormones. Only estrogen inhibition of LG 20 kDa was prevented by simultaneous tamoxifen administration. Simultaneous treatment of gonadectomized hamsters with gonadotrophins and estrogen/androgen did not prevent the LG 20 kDa protein's inhibition. Relative potencies of estrogens (3.6 μg daily dose) were: estradiol-17βdiethylstilbestrol>estrone>estradiol-17, while estriol and chlorotrianisene had no effect. Dexamethasone, progesterone, prolactin, hypothyroid state or adrenalectomy had no effect on LG 20 kDa expression. Western blot studies confirmed the marked repression of LG 20 kDa by estrogen androgen and thyroid hormone and detected the protein in tears of females and gonadectomized hamsters but not in males. Interestingly, among other tissues tested, crossreaction was only seen with the estrogen-repressed 24 and 20.5 kDa major male-specific secretory proteins of hamster submandibular glands (SMG) which were previously reported by us. This strongly indicated that the LG and SMG proteins are products of the same or closely related genes. A possible role for these hamster sex-specific LG and SMG major secretory proteins in olfactory communication is suggested.     

Marked sexual dimorphism of lacrimal gland peroxidase in hamster: repression by androgens and estrogens

Peroxidase secreted in tears by the lacrimal glands is a marker of secretory activity of these glands and is believed to have an antimicrobial function. We report for the first time a marked sex difference in lacrimal gland (LG) peroxidase in hamsters ( approximately 3.4-fold higher activity in females), which is due to an unusual repression by physiological levels of androgens in males. LG peroxidase activity was markedly induced in a time-dependent manner after gonadectomy in males and also females ( approximately 8- and 2-fold, respectively) and was strongly repressed by androgen treatment in a dose- and time-dependent manner. Estrogen treatment of gonadectomized hamsters could also repress LG peroxidase but not below female levels. These repressions by androgens and estrogens were significantly prevented upon co-treatment with their respective receptor antagonists. Western blotting showed that differences in LG peroxidase specific activity, in different sex hormonal states and treatments were due to changes in the levels of peroxidase protein in LG. A tear peroxidase with a clear sex difference suggests that it might also have other novel function(s) in hamster tears.     

Purification, cloning and regulation of a novel acid-lipase-like protein of hamster expressed in lacrimal glands and tears during lactation

We report a novel 48-kDa tear acid-lipase-like protein (TALLP), which is markedly induced in lacrimal glands (LG) and secreted in tears of hamster dams during lactation. TALLP is undetectable in LG and tears of normal hamsters, but is also induced after gonadectomy in both sexes and this is prevented by androgen, estrogen or thyroid hormone treatment. These observations and the obliteration of TALLP upon cessation of lactation suggest that endogenous estrogens (in females) and androgens (in males) completely repress TALLP expression. Purified TALLP is monomeric, contains approximately 18% N-glycosylation and several pI isoforms. TALLP expression was tissue-specific and immunolocalized in LG acinar cells. The cDNA deduced amino-acid sequence of TALLP precursor (398 residue, containing a 19 residues signal-peptide) showed only 43-48% identity with all known mammalian acid-lipases, including even those of other rodents, suggesting that TALLP is a prototype of a new category, within the acid-lipase family. Surprisingly, although the catalytic triad residues and other sequence features important for lipolytic activity are conserved in TALLP, it has no detectable lipase activity. However, TALLP binds the polarity sensitive hydrophobic probe, 1-aminoanthracene (K(d)=12 microM). TALLP might have a unique substrate-specificity or a lipid-binding/carrier function in tears of hamster dams. This is the first report of an acid-lipase-like protein secreted in tears of any species. Since TALLP lacks the usual lipase activity, it can be an excellent model to understand better what other structural features in acid-lipases influence their catalytic activity.     

Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains.

trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression.     

Tissue kallikrein mK13 is a candidate processing enzyme for the precursor of interleukin-1beta in the submandibular gland of mice

By using Western blot analysis, high levels of 17.5- and 20-kDa interleukin-1beta (IL-1beta) proteins were detected in the submandibular gland (SMG) of mice. Despite this fact, the amount of pro-IL-1beta protein, a precursor of IL-1beta, with a molecular size of 35 kDa in this tissue was below the detectable level, although strong expression of pro-IL-1beta mRNA was observed. A large amount of 17.5-kDa IL-1beta also appeared in the saliva of mice injected with lipopolysaccharide, suggesting that this IL-1beta is a secretory form produced by the SMG. The protein for IL-1beta-converting enzyme, a processing enzyme for pro-IL-1beta, was expressed only at a low level in the SMG as compared with its level in various epithelial tissues or lipopolysaccharide-stimulated macrophages. On the other hand, mK1, mK9, mK13, and mK22, members of the kallikrein family, were detected strongly in the SMG but not in other tissues. By incubation with mK13, but not with mK1, mK9, or mK22, the 35-kDa pro-IL-1beta was cleaved into two major products with molecular masses of 17.5 and 22 kDa, and production was inhibited by phenylmethylsulfonyl fluoride, a serine protease inhibitor, but not by IL-1beta-converting enzyme inhibitors. A peptide segment corresponding to amino acid residues 107-121 of mouse pro-IL-1beta (107WDDDDNLLVCDVPIR) was cleaved by incubation with mK13, generating two peptides, 107WDDDDNL and 114LVCDVPIR. Therefore, kallikrein mK13 would appear to hydrolyze pro-IL-1beta between its Leu113 and Leu114 residues. The results of immunohistochemistry and an autonomic therapy experiment showed that IL-1beta and kallikrein mK13 were co-localized in the secretory granules of granular convoluted tubular cells. Our present results thus suggest kallikrein mK13 is a plausible candidate for the processing enzyme for pro-IL-1beta in the SMG of mice.     

Expression and functional analysis of beta-adrenoceptor subtypes in rabbit submandibular gland

beta-Adrenoceptors (beta-ARs) mediate important physiological functions in salivary glands. Here we investigated the expression and function of beta-AR subtypes in rabbit submandibular gland (SMG). Both beta(1)- and beta(2)-ARs, but not beta(3)-AR, were strongly expressed in rabbit SMG. beta(1)-AR proteins were widely expressed in acinar and ductal cells whereas beta(2)-AR proteins were mainly detected in ductal cells. A [(3)H]-dihydroalprenolol binding assay revealed that beta-AR B(max) was 186+/-11.9 fmol/mg protein and K(d) was 2.71+/-0.23 nM. A competitive binding assay with CGP 20712A, a beta(1)-AR antagonist, indicated that the proportion of beta(1)-AR and beta(2)-AR was 71.9% and 28.1%, respectively. Gland perfusion with the beta-AR agonist isoproterenol induced a significant increase in saliva secretion which was abolished by pretreatment with the non-selective beta-AR antagonist propranolol. Pretreatment with beta(1)- or beta(2)-AR selective antagonists, CGP 20712A or ICI 118551, diminished isoproterenol-induced increase in saliva secretion by 71.2% and 28.8%, respectively. The expression of alpha-amylase mRNA was significantly stimulated by isoproterenol, which was eliminated by propranolol and CGP 20712A. Perfusion with isoproterenol decreased alpha-amylase protein storage in SMG and increased alpha-amylase activity in saliva. These alterations became less significant after pretreatment with propranolol and CGP 20712A. Our results suggest that both beta(1)- and beta(2)-ARs are expressed in rabbit SMG. beta(1)-AR is the predominant subtype and may play an important role in regulating saliva and alpha-amylase secretion.     

Androgen repression of the production of a 29-kilodalton protein and its mRNA in the rat ventral prostate

The regression of the ventral prostate, after a rat is deprived of androgens by castration, is accompanied by a marked decrease in the prostate's ability to synthesize RNA and major proteins. Surprisingly, in vitro translation of prostate RNA, isolated from rats 2 days after castration, detects four proteins with Mr of approximately 29,000, 37,000, 46,000, and 49,000 whose message levels increased 4- to 12-fold compared to results from normal rats. According to cDNA dot hybridization analysis, the increase after castration in the level of the 29-kDa protein-mRNA (per unit amount of DNA) was reversed within 6 h by androgen treatment of castrated rats. In contrast, the level of a mRNA in male rat liver, which hybridized to a cloned probe for the prostate 29-kDa protein-mRNA was reduced by castration and increased by androgen treatment. During an in vitro incubation, the ventral prostates of normal rats were much less efficient than the prostates of rats castrated 2 days earlier in synthesizing a 29-kDa protein. Despite the fact that androgenic manipulation of rats induced very rapid and significant changes in the production of the 29-kDa protein and in the level of its mRNA, the cellular level of this protein in the prostate, as determined by radioimmunoassay, was maintained at near normal values throughout the 2-week experimental period. Thus, the prostate appears to have a mechanism, based on androgen repression of certain genes, to maintain the cellular levels of the 29-kDa protein and possibly other structurally or functionally important proteins during both the periods of androgen-dependent growth and the castration-induced regression. The loss of such a regulatory mechanism may result in androgen-independent abnormal prostate growth.     

Regulation and ligand-binding specificities of two sex-specific bile acid-binding proteins of rat liver cytosol

Rat liver cytosolic proteins were photoaffinity labeled with the synthetic steroid [3H]methyltrienolone in order to identify and characterize hepatic proteins that may participate in the intracellular binding and transport of steroid hormones and other sterols. A male-specific and a female-specific sterol-binding protein (SBP) that migrated to the 4 S region of a sucrose gradient and had similar molecular weights (male-specific 34-kDa protein (SBP34), female-specific 31-kDa protein (SBP31] were thus identified. Experiments were undertaken to determine the biochemical basis for the sex-specific expression of these two proteins. In vivo hormonal manipulations established that the female-specific expression of SBP31 could, in part, be accounted for by the suppressive effects of androgen on SBP31 levels in male rats. In contrast, androgen stimulated expression of the male-specific SBP34, while estrogen and the estrogen-regulated continuous plasma growth hormone profile that is characteristic of adult female rats were suppressive toward this protein. Unlike several other androgen-dependent hepatic proteins, however, SBP34 did not require an intact pituitary for androgen-stimulated expression, nor was its expression stimulated by the intermittent pulses of plasma growth hormone that are characteristic of adult male rats. SBP34 and SBP31 were not induced but were suppressed to various extents by dexamethasone, phenobarbital, and clofibrate, drugs that are known to induce other hepatic proteins involved in steroid binding and metabolism. Competition experiments revealed that SBP31 has a relatively broad ligand specificity, with significant competition for [3H]methyltrienolone binding exhibited by bile acids (chenodeoxycholic acid and lithocholic acid) and a range of steroid hormones (progesterone, estradiol, testosterone, and 5 alpha-dihydrotestosterone) when present in the low micromolar range. No binding was detected with this protein toward cholesterol, triamcinolone acetonide, 5 alpha-androstan-3 alpha,17 beta-diol, cholic acid, and deoxycholic acid. In contrast, SBP34 exhibited greater binding specificity, with competition for [3H]methyltrienolone binding observed only with primary bile acids (cholic acid and chenodeoxycholic acid) and their metabolites (deoxycholic acid and lithocholic acid). On the basis of these binding specificities and the relatively high concentration of bile acids found in the liver, it is proposed that SBP31 and SBP34 function in the intracellular binding and/or transport of bile acids.     

Expression and characteristics of vanilloid receptor 1 in the rabbit submandibular gland

Vanilloid receptor 1 (VR1) is a polymodal receptor originally found in sensory neurons of the central nervous system. Recent evidence indicates that VR1 is also expressed in non-neuronal tissues. We report here endogenous expression of VR1 in rabbit submandibular gland (SMG) and its possible role in regulating saliva secretion based on: (i) the expression of VR1 mRNA and protein detected in SMG; (ii) VR1 was mainly localized in the basolateral membrane of duct cells and the cytoplasm of acinar cells and also in cytoplasm of primary cultured neonatal rabbit SMG cells; (iii) stimulation of neonatal rabbit SMG cells with capsaicin induced a significant increase in intracellular calcium, and capsazepine, a VR1 antagonist, abolished this increase; (iv) infusion of capsaicin via the external carotid artery to isolated SMG increased saliva secretion of the gland. These findings indicated that VR1 was expressed in SMG and appeared to play an important role in regulating saliva secretion.     

Developmental and hormonal regulation of specific proteins in mouse vas deferens and seminal vesicle.

This paper is concerned with hormonal regulation of the developmental pattern of major proteins of the mouse vas deferens (mouse vas deferens protein: MVDP, 34.5 kD) and seminal vesicle (15.5, 120 and 140 kD) whose expression is regulated by testosterone at adulthood. The ontogeny of these proteins, studied by SDS-polyacrylamide gel electrophoresis, appeared to be uncoordinated. MVDP was not accumulated until animals were 20 days old and its concentration increased sharply from 20 to 30 days of age. In seminal vesicle, the 15.5 kD protein did not accumulate before day 30 whereas 120 and 140 kD proteins appeared and accumulated between 30 and 40 days. In 30-day-old mice castrated at birth or treated with cyproterone acetate over 29 days, MVDP levels were not abolished and were similar to those measured in 20-day-old males. Testosterone administration, from 1 to 10 days of age, did not induce precocious expression of MVDP. These results suggest that the neonatal expression of MVDP is independent of androgens. In seminal vesicle, the first expression of the 3 proteins studied was dependent upon testicular androgens as shown by neonatal castration and injection experiments. The marked increase in the levels of the 4 proteins studied, during sexual maturation, was not associated with quantitative or qualitative changes in tissular androgen concentrations, suggesting that other factors may be necessary for protein expression. Whereas thyroxine may induce a precocious accumulation of MVDP, prolactin had no stimulatory effect on the accumulation of proteins from vas deferens and seminal vesicle. The results suggest that during sexual maturation gene activation by androgens was progressive.     

Characterization of small-molecular-mass guanine-nucleotide-binding regulatory proteins in insulin-secreting cells and PC12 cells.

The distribution of ras-related small-molecular-mass guanine-nucleotide-binding regulatory proteins (SMG) of two insulin-secreting cell lines, RINm5F and HIT-T15, and of a catecholamine-secreting cell line, PC12, have been studied using different techniques. About ten such proteins were detected by [32P]GTP binding after two-dimensional gel electrophoresis and transfer to nitrocellulose membranes. In insulin-secreting cells, rho protein(s) that cannot be detected with the GTP-binding technique were identified by ADP ribosylation with Clostridium botulinum C3 exoenzyme. After subcellular fractionation, SMG displayed specific distributions. The insulin-secreting cell line RINm5F and the catecholamine-secreting cell line PC12 expressed a similar set of these proteins with analogous localization. [32P]GTP binding analysis revealed that at least seven SMG were associated with the secretory granule enriched fraction of RINm5F cells and with the fraction containing dense secretory granules from PC12 cells, proteins of 27 (pI 5.4), 23 (pI 6.8) and 25 kDa (pI 6.7) being the most abundant. These proteins were present in a highly purified granule fraction of a solid rat insulinoma. The 23 kDa (pI 6.8) and 25 kDa (pI 6.7) proteins, but not the protein migrating at 27 kDa (pI 5.4), were detected in the corresponding fraction from HIT-T15 cells. A monoclonal antibody directed against smg25A/rab3A recognized the SMG in secretory granules migrating at 25 kDa (pI 6.7) and 27 kDa (pI 5.4). This antibody also revealed the presence of such protein(s) in homogenates of rat pancreatic islets. During stimulation of insulin secretion of either intact or permeabilized cells, there was no detectable redistribution to the cytosol or to the plasma membrane of the major proteins located on secretory granules. In view of the invariable presence of at least two of the SMG in granules of secretory cells, these proteins are good candidates for regulation of hormone secretion.     

Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1

We recently reported that antibody against purified P450 3A1 (P450p) recognizes two electrophoretically distinct proteins (50 and 51 kDa) in liver microsomes from male and female rats, as determined by Western immunoblotting. Depending on the source of the liver microsomes, the 51-kDa protein corresponded to 3A1 and/or 3A2 which could not be resolved by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. The other protein (50 kDa) appears to be another member of the P450 IIIA gene family. Both proteins were markedly intensified in liver microsomes from male or female rats treated with pregnenolone-16α-carbonitrile, dexamethasone, troleandomycin, or chlordane. In contrast, treatment of male or female rats with phenobarbital intensified only the 51-kDa protein. Treatment of male rats with Aroclor 1254 induced the 51-kDa protein, but suppressed the 50-kDa form. In addition to their changes in response to inducers, the 50- and 51-kDa proteins also differed in their developmental expression. For example, the 50-kDa protein was not expressed until weaning (3 weeks), whereas the 51-kDa protein was expressed even in 1-week-old rats. At puberty (between weeks 5 and 6), the levels of the 50-kDa and 51-kDa proteins markedly declined in female but not in male rats, which introduced a large sex difference (male > female) in the levels of both proteins. Changes in the level of the 51-kDa protein were paralleled by changes in the rate of testosterone 2β, 6β-, and 15β-hydroxylation. In male rats, the marked increase in the levels of the 50-kDa protein between weeks 2 and 3 coincided with a three- to four fold increase in the rate of testosterone 2β-, 6β-, and 15β-hydroxylation, which suggests that the 50-kDa protein catalyzes the same pathways of testosterone oxidation as the 51-kDa protein. However, this developmental increase in testosterone oxidation may have resulted from an activation of the 51-kDa 3A protein. These results indicate that the two electrophoretically distinct proteins recognized by antibody against P450 3A1 are regulated in a similar but not identical manner, and suggest that the 51-kDa 3A protein is the major microsomal enzyme responsible for catalyzing the 2β-, 6β-, and 15β-hydroxylation of testosterone.     

Innate resistance to herpes simplex virus infection. Human lymphocyte and monocyte inhibition of viral replication

Peripheral blood monocytes and lymphocytes isolated from most humans are resistant to HSV infection in vitro. Viral replication is inhibited very early in the cycle, prior to the onset of alpha-protein synthesis; no viral protein or DNA synthesis is detectable even up to 1 week later. The enhanced expression of two 62-kDa and 57-kDa cellular proteins, however, is induced in the lymphocyte population within 3 to 5 h after infection. A 30-kDa protein is induced in the monocyte population immediately after infection. The induced expression of 62-kDa and 57-kDa lymphocyte proteins appears to be virus-mediated because: a) HSV and pseudorabies virus (although not vaccinia virus) induce the expression of 62-kDa and 57-kDa proteins, b) heat shock or exposure of lymphocytes to uninfected cell extracts does not induce expression of either protein, c) 62-kDa protein is not induced in lymphocytes stimulated with a mitogenic concentration of PHA. UV-inactivated HSV induces expression of 62-kDa and 57-kDa proteins in a manner similar to that observed with untreated virus. In contrast, expression of 30-kDa monocyte protein is induced nonspecifically by either uninfected cell extracts or cell extracts containing virus. Sixty-two-kilodalton and 57-kDa protein induction appears to be a marker for human lymphocytes that express profound intracellular resistance to infection with HSV. Induced expression of these proteins occurs only in lymphocytes that inhibit viral replication very early in the growth cycle, prior to the onset of alpha-protein synthesis. Expression of 62-kDa and 57-kDa proteins is not induced in lymphocytes that are permissive or partially permissive to infection with HSV.