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M Kashimata

Publications and source records attributed to M Kashimata.

At least 19 recordsLinked to original sources
PubMed2000

The ERK-1/2 signaling pathway is involved in the stimulation of branching morphogenesis of fetal mouse submandibular glands by EGF.

We have previously reported that epidermal growth factor (EGF) stimulates branching morphogenesis of the fetal mouse submandibular gland (SMG) (M. Kashimata and E. W. Gresik, 1997, Dev. Dyn. 208, 149-161) and that the EGF receptor (EGFR) is localized principally, if not exclusively, on the epithelial components of the fetal SMG (E. W. Gresik, M. Kashimata, Y. Kadoya, R. Mathews, N. Minami, and S. Yamashina, 1997, J. Histochem. Cytochem. 45, 1651-1657). The EGFR is a receptor tyrosine kinase, and after binding of its ligand, it triggers several intracellular signaling cascades, among them the one activating the mitogen-activated protein kinases (MAPK) ERK-1/2. Here we investigated whether EGF utilizes the ERK-1/2 signaling cascade to stimulate branching morphogenesis in the fetal mouse SMG. SMG rudiments were collected as matched pairs at E14, E16, and E18 (E0 = day of vaginal plug); placed into wells of defined medium (BGJb); and exposed to EGF for 5 or 30 min or to medium alone (controls). By Western blotting we found that EGF induced the appearance of multiple bands of phosphotyrosine-containing proteins, including bands at 170 kDa and 44 kDa/42 kDa, presumably corresponding to the phosphorylated forms of EGFR and ERK-1/2, respectively. Other blots showed the specific appearance of the phosphorylated EGFR and of phospho-ERK-1/2 in response to EGF. Immunohistochemical staining for phosphotyrosine increased at the plasma membrane after EGF stimulation for 5 or 30 min. Diffuse cytoplasmic staining for MEK-1/2 (the MAPK kinase that activates ERK-1/2) increased near the cell membrane after EGF stimulation. Phospho-ERK-1/2 was localized in the nuclei of a few epithelial cells after EGF for 5 min, but in the nuclei of many cells after EGF for 30 min. PD98059, an inhibitor of phosphorylation and activation of MEK-1/2, by itself inhibited branching morphogenesis and, furthermore, decreased the stimulatory effect of EGF on branching. Western blots confirmed that this inhibitor blocked phosphorylation of ERK-1/2 in fetal SMGs exposed to EGF. These results show that components of the ERK-1/2 signaling cascade are present in epithelial cells of the fetal SMG, that they are activated by EGF, and that inhibition of this cascade perturbs branching morphogenesis. However, EGF did not cause phosphorylation of two other MAPKs, SAPK/JNK or p38MAPK, in fetal SMGs. These results imply that the ERK-1/2 signaling is responsible, at least in part, for the stimulatory effect of EGF on branching morphogenesis of the fetal mouse SMG.

Animals↗
PubMed1998

The EGF system in fetal development.

Immunohistochemical localization of the epidermal growth factor receptor (EGFR) disclosed a wide distribution of this protein in diverse fetal tissues. This review focuses on some of the vast work on the expression and operation of the EGFR and its principal ligands in fetal organogenesis.

Animals↗
PubMed1997

Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the alpha6-integrin subunit.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) regulate branching morphogenesis of fetal mouse submandibular gland (SMG) rudiments in vitro. The EGF system (EGF, TGF-alpha, and their shared receptor, EGFR) also regulates expression of integrins and their ligands in the extracellular matrix. We show here that inhibition of EGFR tyrosine-kinase activity by a tyrphostin retards in vitro development of SMGs. Using total RNA isolated from pooled SMGs taken from intact mouse fetuses, mRNA transcripts for EGF, TGF-alpha, and EGFR were detected by reverse transcription-polymerase chain reaction (RT-PCR), and age-dependent variations in the levels of these mRNA were quantitatively determined by nuclease protection assays. These findings suggest that the EGF system is operative in the in vivo development of this gland. alpha6-Integrin subunit was localized by immunofluorescence at the basal surface of epithelial cells. Branching morphogenesis of cultured SMG rudiments was inhibited by anti-alpha6 antibodies. Synthesis of alpha6-subunit in cultured SMGs, detected by metabolic labeling and immunoprecipitation, was increased by EGF and drastically reduced by tyrphostin. RT-PCR revealed that mRNAs for alpha6- and beta1- and beta4-integrin subunits are expressed at all ages between embryonic day 13 and postnatal day 7. These findings suggest that 1) the EGF system is a physiologic regulator of development of fetal mouse SMG, and 2) one mechanism by which it acts may be by regulating expression of integrins, which in turn control interaction of epithelial cells with the extracellular matrix.

Animals↗
PubMed1997

Expression of epidermal growth factor receptor in fetal mouse submandibular gland detected by a biotinyltyramide-based catalyzed signal amplification method.

Branching morphogenesis of the fetal mouse submandibular gland (SMG) can be modulated in vitro by stimulation or inhibition of the epidermal growth factor receptor (EGFR). Because the mRNAs for EGF and EGFR are detectable in RNA of SMG rudiments isolated directly from fetuses, the EGF system probably operates physiologically as a regulator of SMG morphogenesis. However, neither EGFR protein nor its precise cellular localization has been characterized in the fetal SMG. Here we show EGFR protein in fetal mouse SMG by immunoprecipitation, affinity labeling, ligand-induced autophosphorylation, and immunohistochemistry. SMGs from E16 fetuses (day of vaginal plug = E0) were labeled with [35S]-cysteine/methionine and homogenized. After addition of specific antibody to EGFR, the immunoprecipitate was isolated, resolved by polyacrylamide gel electrophoresis, and detected by autoradiography. A single band of 170 kD was detected, corresponding to the EGFR protein. Affinity labeling with [125I]-EGF of the membrane fraction of E18 SMG also revealed a prominent band at 170 kD, showing that this EGFR protein can bind specifically to its ligand. Incubation of SMG membranes from E18 fetuses with EGF in the presence of [gamma-32P]-ATP, followed by immunoprecipitation with anti-phosphotyrosine antibody also showed a single band at 170 kD, demonstrating autophosphorylation of the EGFR in response to binding of its ligand. Immunohistochemical localization of the cellular sites of EGFR in the fetal SMG required use of a catalyzed signal amplification procedure, with biotinyltyramide as the amplifying agent. EGFR was localized predominantly, if not exclusively, in cell membranes of epithelial cells of the rudiment, whereas staining of mesenchymal cells was equivocal. Staining was strongest on duct cells, and weak on cells of the end-pieces. These findings clearly show that a functional EGFR protein is expressed in fetal SMG chiefly, if not exclusively, on epithelial cells.

Affinity Labels↗
PubMed1996

Contemporary approaches to the study of salivary gland morphogenesis.

The submandibular gland of the mouse and rat serves as an important model to study epitheliomesenchymal interactions, since it can be studied both in vivo and in vitro. Previous work has established that the extracellular matrix, and especially the basal lamina, play vital roles in the development of this gland. More recent studies have indicated that integrins and several oncogene-related products, including components of the epidermal growth factor system, also play important roles in this gland's development.

Animals↗
PubMed1994

Immunohistochemical analysis of EGF in epiphyseal growth plate from normal, hypophysectomized, and growth hormone-treated hypophysectomized rats.

Epiphyseal growth plate cartilages from the proximal tibia of normal, hypophysectomized, and growth hormone (GH)-treated hypophysectomized rats were subjected to immunohistochemistry for detection of epidermal growth factor (EGF). In the normal growth plate, EGF was distributed mainly in the proliferative zone. Hypophysectomy resulted in considerable atrophy of the chondrocytes and the cartilage matrix (a decreased number of mature-type chondrocytes and a decreased ratio of proliferating to hypertrophic chondrocytes) and a significant diminution of EGF immunoreactivity. Treatment with GH reversed these effects of hypophysectomy, causing an increased thickness of the growth plate and EGF-reactive sites in all chondrocyte layers. The most intense immunostaining for EGF, however, was frequently seen in the nuclei of chondrocytes with flattened appearance. It appears that EGF could be incorporated or synthesized in chondrocytes having marked mitogenic activity. The present results, taken with previous data on EGF involvement in growth of cartilaginous tissue in vivo and in vitro, strongly suggest that EGF-immunoreactive chondrocytes are involved in cartilage proliferation and growth under the specific influence of GH.

Animals↗
PubMed1994

Developmental changes in and hormonal modulation of epidermal growth factor concentration in the rat submandibular gland.

Developmental changes in the hormonal effects on the concentration of epidermal growth factor (EGF) in the rat submandibular gland were investigated. The level of EGF in the gland gradually increased with age from 4 up to 8 weeks of age; thereafter it increased markedly, reaching a plateau level at 12 weeks of age in both male and female rats. A significant sex difference in EGF levels was observed between 8 and 14 weeks, the level in the males being approximately twice as high as that in the females at 12 and 14 weeks of age. Castration of male rats decreased EGF to about the same level as that of control females. Treatment of castrated rats with testosterone propionate (TP) restored EGF to the levels in control male rats. Ovariectomy and/or administration of oestradiol-17 beta to ovariectomized rats had no apparent effect on EGF concentration. These findings indicate that the sex difference in EGF concentration can be attributed to the level of endogenous androgens. In addition, hypophysectomy of male rats caused a remarkable decrease in submandibular gland EGF to about 7% of the normal level. This reduction was significantly, although not completely, restored by the administration of TP, triiodothyronine (T3) or GH. Moreover, giving TP with T3 or with GH or both together had additive effects on the increase in EGF levels in hypophysectomized rats. These results provide evidence that EGF in the submandibular gland is regulated multihormonally by at least TP, T3 and GH.

Aging↗
PubMed1991

Purification and tissue distribution of rat epidermal growth factor.

1. Two forms of rat epidermal growth factor, EGF-I and EGF-II, were purified to homogeneity from male rat submandibular glands. 2. The mol. wts of EGF-I and -II were estimated to be 5200 and 5400, respectively, both of them having an apparent biological activity. 3. The antiserum against EGF-II strongly cross-reacted with EGF-I; however, it did so only slightly with mouse or human EGF. 4. EGF was detected by radioimmunoassay in various tissues of male and female rats, and the concentrations of rat EGF in the submandibular gland, parotid gland, sublingual gland, and liver were significantly higher in the male than in the female.

Animals↗
PubMed1990

[Sex differences and regulatory mechanism of epidermal growth factor receptor in the rat liver].

The number of epidermal growth factor (EGF) receptor in rat hepatic membranes was about 2-hold higher in adult male than in adult female rats. Castration of adult males slightly decreased the EGF receptor number. Castration of neonatal males decreased the number of EGF receptors when they reached sexual maturity. This decrease was restored by the combination of neonatal and pubertal treatments with testosterone. Hypophysectomy caused a marked decrease in the number of EGF receptors in the male animals, and this decrease was not restored by either testosterone or triiodothyronine administration. Continuous administration of human growth hormone (hGH) with an osmotic minipump to normal males reduced the EGF receptor number. In contrast, intermittent administration of hGH twice a day (every 12 hr) to hypophysectomized males and/or normal females significantly increased the EGF receptor number. These results indicate that the number of EGF receptors in rat hepatic membranes is regulated by the secretory rhythm of GH in the pituitary, which may be "imprinted" by neonatal androgen.

Animals↗
PubMed1990

[Properties of N-acetyl-beta-glucosaminidase in the masseter muscle of the mouse].

The present study was undertaken to clarify various characteristics of N-acetyl-beta-glucosaminidase (NAG) in the masseter muscle of the mouse. The pH optimum of NAG in tissue homogenate was 4.3, and Km value for p-nitrophenyl-N-acetyl-beta-glucosaminide was 0.98 mM. By means of isoelectric focusing. NAG in the masseter muscle was separated into four enzymes (NAG I-IV). The isoelectric points were: I, 4.9; II, 6.4; III, 7.2 and IV, 8.7. The pH optima of NAG I-IV ranged from 4.2 to 4.4, and Km values from 0.61 to 0.83 mM. The molecular weights of I, II, III and IV were 135,000, 120,000, 135,000 and 110,000, respectively. Studies on heat stability showed that NAG I and IV were slightly labile as compared with II and III. The activities of NAG I-IV were dose dependently inhibited by both N-acetyl-glucosamine and N-acetyl-galactosamine. Both Ag and Hg2+ ions caused a marked inhibition on activities of NAG I-IV.

Acetylglucosaminidase↗
PubMed1990

Effects of sialoadenectomy and epidermal growth factor administration on the duodenum of adult mice.

Sialoadenectomy (removal of the submandibular glands) of adult male and female mice caused significant or apparent decreases in duodenal weight, duodenal protein, DNA and RNA contents, and alkaline phosphatase activity. These decreased levels observed in sialoadenectomized mice were completely restored to or rather increased over the control levels by epidermal growth factor (EGF) administration. There was no appreciable difference in body weight between the control and sialoadenectomized mice. These results strongly suggest that salivary EGF secreted from the submandibular gland plays a role in the maintenance of growth and function(s) of the adult mouse duodenum.

Alkaline Phosphatase↗
PubMed1989

Characteristics of sialidase in the rat salivary glands.

Using 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc) as substrate, we measured sialidase activity in the salivary glands and other organs of the rat. The pH optima of salivary gland sialidase were between 4.0 and 4.5, which were similar to those of the enzyme in the brain, liver and kidney. Among the salivary glands, the submandibular one showed the highest sialidase activity followed by the parotid and the sublingual glands. However, sialidase activity in these glands was lower when compared with the activity in the brain, liver and kidney. From the subcellular distribution study, salivary gland sialidase was found to be mainly localized in the lysosomes. The pH optima of the lysosomal sialidase of the salivary glands were between 4.0 and 4.5; and Km values for 4MU-NeuAc approximately 0.09 mmol/l. In the submandibular and parotid glands, a soluble sialidase with a different pH optimum (5.5) and Km value (0.25 mmol/l) was also detected.

Animals↗
PubMed1989

Differential secretory rhythm of growth hormone controls the number of hepatic epidermal growth factor receptors in the rat.

The effect of human GH (hGH) on hepatic epidermal growth factor (EGF) receptors in the rat was investigated. Continuous administration of hGH through an osmotic minipump, mimicking the female pattern of GH secretion, to normal male rats reduced the binding of 125I-labelled EGF to hepatic membranes to the normal female levels. The same treatment of hGH applied to hypophysectomized males had no apparent effect on EGF binding. Intermittent s.c. administration of hGH twice a day (every 12 h), mimicking the male pattern of GH secretion, to hypophysectomized male and/or normal female rats, caused a significant increase in EGF binding to the levels of normal male rats. Scatchard analysis of the binding data clearly showed that the change in EGF binding was due to a change in the number of EGF receptors. The results on the affinity labelling and phosphorylation of EGF receptors were in good agreement with those showing differences in the number of EGF receptors among the experimental groups. These results indicate that the number of hepatic EGF receptors in the rat is regulated by the differential secretory rhythm of pituitary GH between the sexes.

Animals↗
PubMed1989

[Sialidase in rat salivary glands. Characteristics of soluble sialidase in salivary glands and other tissues].

In this study, we elucidated some characteristics of soluble sialidases in the rat salivary glands, brain, liver and kidney. 1) Soluble sialidases in the submandibular and parotid glands were inactivated by about 75 and 60%, respectively, and the enzymes in the brain, liver and kidney were also inactivated by 16-48%, when preincubated at 40 degrees C for 1h. When preincubated at 60 degrees C, the enzymes in all tissues were completely inactivated within 10 min. 2) Ca2+ ion in low concentrations tended to activate soluble sialidases in both the submandibular and parotid glands, but had no apparent effect on the enzymes in the brain, liver and kidney. The enzyme in the submandibular gland tended to be activated also by low concentrations of Mg2+. Both Cu2+ and Hg2+ ions caused a marked inhibition on the soluble sialidases in all tissues. 3) The molecular weight of soluble sialidases in the submandibular and parotid glands was determined to be about 68,000 and 46,000, respectively, by means of Sephadex G-200 gel filtration. The molecular weight of the enzymes in the brain and liver was almost similar to that of the enzyme in the parotid gland. In the kidney, soluble sialidase was separated into two enzymes, one having high (more than 500,000) and the other having low (about 46,000) molecular weight. 4) By isoelectric focusing analysis, three isozymes were detected in the submandibular gland and brain sialidases and two isozymes detected in the enzymes of other tissues. The isoelectric point of the major isozymes in the submandibular and parotid glands was 6.4 and 6.9 respectively. In the brain, liver and kidney sialidases, the isozymes with isoelectric point in the range of 4.4 to 6.7 were detected.

Animals↗