Characterization of mouse tissue kallikrein 5

Mouse tissue kallikreins (Klks) are members of a large, multigene family consisting of 37 genes, 26 of which can code for functional proteins. Mouse tissue kallikrein 5 (Klk5) has long been thought to be one of these functional genes, but the gene product, mK5, has not been isolated and characterized. In the present study, we prepared active recombinant mK5 using an Escherichia coli expression system, followed by column chromatography. We then determined the biochemical and enzymatic properties of purified mK5. mK5 had trypsin-like activity for Arg at the P1 position, and its activity was inhibited by typical serine protease inhibitors. mK5 degraded gelatin, fibronectin, collagen type IV, high-molecular-weight kininogen, and insulin-like growth factor binding protein-3. Our data suggest that mK5 may be implicated in the process of extracellular matrix remodeling.     

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.     

Characterization of mouse glandular kallikrein 24 expressed in testicular Leydig cells

Mouse kallikrein 24 is thought to encode a functional serine protease belonging to the mouse glandular kallikrein gene family. Preliminary results suggest that this kallikrein may play a role in testis function in adult mice. In order to obtain insights into its physiological functions, we undertook molecular and biochemical analyses of this enzyme. We cloned a cDNA for kallikrein 24 from the adult mouse testis cDNA library. Kallikrein 24 was expressed in the kidney, submandibular glands, ovary, epididymis, and testis of the mouse. In the testis, kallikrein 24 mRNA was detectable at 4 weeks of postnatal development, and became more prominent thereafter. The kallikrein 24 gene was expressed exclusively in the Leydig cells of adult mice. When Leydig cells isolated from a 2-week-old mouse testis were cultured in the presence of testosterone, kallikrein 24 expression was induced. Active recombinant enzyme showed trypsin-like specificity, favorably cleaving Arg-X bonds of synthetic peptide substrates. The enzymatic activity was strongly inhibited by typical serine protease inhibitors. Mouse kallikrein 24 degraded casein, gelatin, fibronectin and laminin. These results suggest that the enzyme may play a role in the degradation of extracellular matrix proteins in the interstitial area surrounding the Leydig cells of the adult mouse testis. The present findings should contribute to future physiological studies of this mouse testis protease.     

Expression of nerve growth factor and its receptors NTRK1 and TNFRSF1B is regulated by estrogen and progesterone in the uteri of golden hamsters

Experiments were conducted using female golden hamsters to identify the presence of nerve growth factor (NGF) and its receptors NTRK1 and TNFRSF1B in the uteri of female animals and regulation on their expression by estrogen and progesterone. NGF and its receptor NTRK1 were immunolocalized to luminal epithelial cells, glandular cells, and stromal cells. TNFRSF1B was immunolocalized in luminal epithelial and glandular cells, with no staining found in stromal cells of the uterine horns of normal cyclic golden hamsters. Strong immunostaining of NGF and its receptors NTRK1 and TNFRSF1B was observed in uteri on the day of proestrus as compared to the other stages of the estrous cycle. Results of immunoblot analysis of NGF revealed that there was a positive correlation between uterine NGF expression and plasma concentrations of estradiol-17beta. To clarify the effects of estrogen and progesterone on NGF, NTRK1, and TNFRSF1B expression, adult female golden hamsters were ovariectomized and treated with estradiol-17beta and/or progesterone. Immunoblot analysis and immunohistochemistry indicated that estradiol-17beta stimulated expression of NGF and its two receptors in the uterus. Treatment with progesterone also increased NGF and NTRK1 expression in the uterus. However, no additive effect of these steroids on expression of NGF and its receptors was observed. Changes in uterine weights induced by estradiol-17beta and/or progesterone showed the same profile with that of NGF, suggesting that a proliferative act of NGF may be involved in uterine growth. These results suggest that NGF may play important roles in action of steroids on uterine function.     

Effects of luteinizing hormone and follicle-stimulating hormone and insulin-like growth factor-I on aromatase activity and P450 aromatase gene expression in the ovarian follicles of red seabream,Pagrus major

To clarify the mechanism of estradiol-17beta production in the ovarian follicle of red seabream, in vitro effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and insulin-like growth factor (IGF-I) on aromatase activity (conversion of testosterone to estradiol-17beta) and cytochrome P450 aromatase (P450arom) mRNA expression in ovarian fragments of red seabream were investigated. Of the growth factors used in the present study, only IGF-I stimulated both aromatase activity and P450arom gene expression in the ovarian fragments of red seabream. LH from red seabream pituitary, but not FSH, stimulated both aromatase activity and P450arom gene expression. IGF-I slightly enhanced the LH-induced aromatase activity and P450arom gene expression. These data and our previous results indicate that LH, but not FSH, stimulates estradiol-17beta production in the ovarian follicle of red seabream through stimulation of aromatase activity and P450arom gene expression and IGF-I enhances the LH-stimulated P450arom gene expression.     

Androgen regulation of the cellular Distribution of the true tissue kallikrein mK1 in the submandibular gland of the mouse.

The kallikrein gene family encodes for at least four different proteases in the mouse submandibular gland (SMG): mK1 (true tissue kallikrein), mK9, mK13, and mK22. These enzymes and many other biologically active proteins are synthesized by the granular convoluted tubule (GCT), a specialized segment of the SMG duct system. The GCT is under multihormonal regulation by androgens, thyroid hormones, and adrenocortical hormones. Androgens suppress synthesis of mK1 in the SMG but enhance expression of the other three kallikreins. We prepared an antibody with limited immunoreactivity for mK1 and used it to examine the effects of androgen status on the distribution of this isozyme in the SMGs of developing and mature mice by immunoperoxidase staining for the light microscope and immunogold labeling for the electron microscope. In prepubertal mice, every immature GCT cell contains mK1, confined to an accumulation of small granules in the subluminal cytoplasm. In mature mice, not every GCT cell contains mK1, and in those cells that do there is considerable intergranular variation in the intensity of staining for mK1. GCT cells containing mK1 are much more abundant in the glands of females than of males, resulting in a peculiar sexually dimorphic mosaic distribution of this isozyme in the mature SMG. Castration of adult males increases the number of GCT cells expressing mK1. Administration of androgen to intact or castrated males or to intact females reduces the number of cells staining for mK1. In all cases, immunogold labeling for mK1 is confined to secretory granules. No fine structural differences were noted between cells that were positively or negatively stained for mK1. Therefore, although GCT cells appear to be composed of a uniform population of cells on the basis of morphology alone, they are not homogeneous in their content of secretory proteins. These results indicate that androgen regulation of GCT cells is more complex than has been appreciated to date.     

Ovarian regulation of endometrial gland morphogenesis and activin-follistatin system in the neonatal ovine uterus

Postnatal development of the ovine uterus between birth and Postnatal Day (PND) 56 involves differentiation of the endometrial glandular epithelium from the luminal epithelium followed by tubulogenesis and branching morphogenesis. Previous results indicated that ovariectomy of ewes at birth did not affect uterine growth or initial stages of endometrial gland genesis on PND 14 but did affect uterine growth after PND 28. Available evidence from a number of species supports the hypothesis that the ovary does not affect endometrial gland morphogenesis in the postnatal uterus. To test this hypothesis in our sheep model, ewes were assigned at birth to a sham surgery as a control or bilateral ovariectomy (OVX) on PND 7. Uteri were removed and weighed on PND 56. Ovariectomy did not affect circulating levels of estradiol-17beta. Uterine weight was 52% lower in OVX ewes. Histomorphological analyses indicated that the thickness of the endometrium and myometrium, total number of endometrial glands, and endometrial gland density in the stratum spongiosum stroma was reduced in uteri of OVX ewes. In contrast, the number of superficial ductal gland invaginations and gland density in the stratum compactum stroma was not affected by ovariectomy. The uteri of OVX ewes contained lower levels of betaA subunit, activin receptor (ActR) type IA, ActRIB, and follistatin protein expression but higher levels of betaB subunit. In the neonatal ovary, follistatin, inhibin alpha subunit, betaA subunit, and betaB subunit were expressed in antral follicles between PNDs 0 and 56. These results led to rejection of the hypothesis that the ovary does not influence endometrial adenogenesis. Rather, the ovary and, thus, an ovarian-derived factor regulates, in part, the coiling and branching morphogenetic stage of endometrial gland development after PND 14 and expression of specific components of the activin-follistatin system in the neonatal ovine uterus that appear to be important for that critical process.     

Expression and enzymatic characterization of recombinant human kallikrein 14

Human kallikrein 14 (KLK14) is a member of the human kallikrein gene family of serine proteases, and its protein, hK14, has recently been suggested to serve as a new ovarian and breast cancer marker. To gain insights into hK14's physiological functions, the active recombinant enzyme was obtained in an enzymatically pure state for biochemical and enzymatic characterizations. We studied its substrate specificity and behavior to various protease inhibitors, and identified candidate physiological substrates. hK14 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. The protease degraded casein, fibronectin, gelatin, collagen type I, collagen type IV, fibrinogen, and high-molecular-weight kininogen. Furthermore, it rapidly hydrolyzed insulin-like growth factor binding protein-3 (IGFBP-3). These findings suggest that hK14 may be implicated in tumor progression in ovarian carcinoma.     

Decidualization induces the expression and activation of an extracellular protease neuropsin in mouse uterus

Uterine decidualization is accompanied by the remodeling of the cell-matrix and cell-cell interactions around the endometrial stromal cells to allow an appropriate invasion of trophoblasts. This remodeling is thought to require the proteolysis of extracellular matrix proteins or cell adhesion molecules; however, the molecular mechanism remains poorly understood. In this study, decidualization induced the expression and activation of an extracellular serine protease neuropsin in the mouse uterus. Although nonpregnant uteri contained little neuropsin, the protein content and enzymatic activity increased markedly and peaked at the midgestational period in pregnant uteri. Neuropsin expression and activity was also upregulated in artificially induced deciduomata but not in nondecidualized pseudopregnant uteri. Neuropsin is the first extracellular protease to show the evident induction of expression and activity by decidualization and might contribute to the remodeling of extracellular components after decidualization.     

Increase in the number of integrinbeta1-immunoreactive monocyte-lineage cells in experimentally-induced adenomyosis in mice

Uterine adenomyosis is a disease in which hyperplastic endometrial stroma and glands invade the myometrium. We have previously demonstrated that hyperprolactinemia leads to the development of adenomyosis in mice. In the present study, a subtracted cDNA library was made by suppression subtractive hybridization to find specific genes that are abundantly expressed in the adenomyotic but not normal tissue in mice. A cDNA fragment of integrinbeta1 (ibeta1) was found in the library, and the expression of the gene product was increased in the adenomyotic uteri at mRNA and protein levels. Intense ibeta1-immunoreactivity was localized on a group of cells dispersing throughout the endometrial stroma. The number of ibeta1-immunoreactive (ibeta1-ir) cells was significantly greater in the uteri of mice with adenomyosis than normal mice. The majority of the ibeta1-ir cells expressed CD14-ir signal, a marker for monocyte-lineage cells, whereas an increase in the number of CD14-ir cells was also evident in the adenomyotic uteri, especially in the ectopic endometrial tissue. Thus, the adenomyotic stromal tissue contained numerous monocyte-lineage cells with higher expression levels of ibeta1, one of their products. The relationship between the increased number of monocyte-lineage cells and the hyperplastic proliferation of endometrial tissues was discussed with a view to understanding the progressive mechanism of adenomyosis.     

Tissue inhibitor of matrix metalloproteinase-3 expression in the mouse uterus during implantation and artificially induced decidualization

During implantation in mice, tissue inhibitor of matrix metalloproteinases-3 is believed to play a key role in inhibiting matrix metalloproteinase activity associated with embryo invasion and tissue remodeling. The first objective of this study was to quantitatively compare the steady-state mRNA levels of tissue inhibitors of matrix metalloproteinases between segments of the mouse uterus undergoing decidualization compared to those that are not during early pregnancy plus oil-induced decidualization. Steady-state tissue inhibitor of metalloproteinase-3 mRNA levels were significantly greater in implantation compared to interimplantation areas on days 6 and 7 of pregnancy and in stimulated compared to nonstimulated uterine horns at 48 and 72 hr after artificial induction of decidualization. Steady-state tissue inhibitor of metalloproteinase-1 mRNA levels were significantly greater in implantation compared to interimplantation areas on days 5-8 of pregnancy and in stimulated compared to nonstimulated uterine horns at 24, 48, and 72 hr after oil stimulation. Therefore, the steady-state mRNA levels of tissue inhibitors of metalloproteinase-1 and -3 increased in the uterus during decidualization. The second objective of this study was to determine if transforming growth factor-beta1 influences tissue inhibitors of metalloproteinase mRNA concentrations in mouse endometrial stromal cells. As determined by Northern blot analyses, transforming growth factor beta1 significantly increased tissue inhibitors of matrix metalloproteinases-1 and -3 mRNA levels in cultured mouse endometrial stromal cells isolated from uteri sensitized for decidualization. On the other hand, interleukin-1, epidermal growth factor, and leukemia inhibitory factor had no effect. The results of this study further characterize the tissue inhibitor of metalloproteinase expression in the uterus during implantation and artificially induced decidualization and the potential control of their expression in the stroma by transforming growth factor.     

In vitro mycotoxin binding to bovine uterine steroid hormone receptors

The mycotoxins, aflatoxin B(1), aflatoxin M(1), aflatoxicol and zearalenone were tested for binding to bovine endometrial estrogen and progestin receptors. Radioinert estradiol-17beta, estrone, testosterone, and cholesterol were evaluated for binding to the estrogen receptor. Zearalenone and aflatoxicol but not aflatoxins B(1) and M(1) competed with estradiol-17beta for the estrogen receptor. The order of binding affinities for the estrogen receptor were zearalenone > estradiol-17beta > estrone > aflatoxicol. The affinity of zearalenone for the estrogen receptor was 2-3 times that of estradiol-17beta. Progesterone, cortisol, radioinert R 5020, and cholesterol were evaluated for binding to the progestin receptor. None of the tested compounds except R 5020 and progesterone competed for the progestin receptor. The significance of aflatoxicol binding to the estrogen receptor is unclear. It is proposed that aflatoxicol binding to the receptor may alter gene expression in target tissues or act at the level of the hypothalamus to inhibit gonadotropin secretion and ovulation. These effects could explain reports of reduced fertility in domestic animals following ingestion of aflatoxin contaminated feedstuffs. It is also suggested that the mechanism of adverse effects on fertility of chronic aflatoxin ingestion in cattle and other livestock should be more thoroughly investigated.     

Expression of rat uterine serine proteinases homologous to mouse implantation serine proteinase 2

Implantation serine protease (ISP) was first identified in the uteri of pregnant mice. It is thought that ISP may have an important role in the initiation of implantation. However, the expression status and detailed functions of ISP remain unclear. In this study, the expression of ISP was investigated in the rat uterus. The analysis of two rat genes registered in GenBank, accession nos. XM_220240 and XM_577076, exhibited high identities to the mouse ISP2 genes, respectively at an mRNA level. We labeled the former as rISP2a and the latter as rISP2b. Using RT-PCR, we found that both genes were expressed in the uterus. Specifically, rISP2a mRNA was detected in the uterus throughout pregnancy, whereas rISP2b mRNA was only expressed in the uterus from day 5 of pregnancy until the end of gestation. Expression of both genes was observed specifically within the endometrial gland epithelium. Furthermore, rISP2a was also observed to be expressed in the fetus and placenta, whereas rISP2b expression was observed in the fetus but not in the placenta. An expressional signal of the rISP2a gene was observed in the spongiotrophoblasts, giant cells and decidual endometrium in the placenta. In the embryo, the ventral specific region was positive in rISP2a and rISP2b gene expression. These findings indicate the possibility that the presently examined genes with high identity to mouse ISP2 may play some role not only during the implantation phase, but also in the development of the placenta and embryo.