Tissue-specific expression in the salivary glands of transgenic mice.

Using a DNA construct, named Lama, derived from the murine parotid secretory protein (PSP) gene, we have obtained salivary gland specific gene expression in transgenic mice. Lama is a PSP minigene and allows analysis of the PSP gene 5' regulatory region by transgenesis. We show here that the regulatory region included in Lama with 4.6 kb of 5' flanking sequence is sufficient to direct expression specifically to the salivary glands. The expression level in the parotid gland is only about one percent of the PSP mRNA level, while that of the sublingual gland is near the PSP mRNA level. This suggests significant differences in the PSP gene regulation in the two glands. In addition, Lama is a secretory expression vector in which cDNAs or genomic fragments can be inserted. We demonstrate that the Lama construct can direct the expression of a heterologous cDNA encoding the C-terminal peptide of human factor VIII to salivary glands and that the corresponding peptide is secreted into saliva.     

Expression of epidermal growth factor in transgenic mice causes growth retardation

The epidermal growth factor (EGF) family of peptides signals through the erbB family of receptor tyrosine kinases and plays important roles in development and tumorigenesis. Both EGF and transforming growth factor (TGF)-alpha only bind to erbB1 and activate it. The precursor of EGF is distinct from that of TGF-alpha in having eight additional EGF-like repeats. We have recently shown that the EGF precursor without these repeats is biologically active and leads to hypospermatogenesis in transgenic mice. Here we present evidence that the growth of transgenic mice widely expressing this engineered EGF precursor is also stunted. These mice were consistently born at half the normal weight and reached almost 80% of normal weight at adulthood. The mechanism involved a reduction of serum insulin-like growth factor-binding protein-3. Chondrocyte development in the growth plate was affected, and osteoblasts accumulated in the endosteum and periosteum. Besides these novel findings on the in vivo effects of EGF on bone development, we observed no sign of tumor formation in our transgenic animals. In contrast to previous reports on TGF-alpha transgenic mice, we show that the biological functions of EGF and TGF-alpha are clearly distinct.     

An immunohistochemical study of secretagogue-induced secretion of epidermal growth factor in the submandibular salivary glands of mice

Routine histological and indirect immunofluorescence techniques were used to examine the histological details of changes in the distribution of epidermal growth factor (EGF) in the submandibular salivary glands of mice during secretion. Comparisons were made bewteen glands of normal mice and those of mice given one of a number of secretagogues at various times prior to sampling. Normal submandibular salivary glands in male mice had an extensive system of convoluted granular tubules (CGT), the cells of which contained EGF. When adrenaline of alpha-phenylephrine was administered, the CGT cells degranulated, and there was a concomitant loss of intracellular EGF-positive immunofluorescence. The excretory ducts were engorged with immunofluorescent material, indicating secretion of EGF into saliva, while the ductal cells themselves remained EGF-negative. The degranulation response could be blocked by phentolamine, but not by propranolol, and no changes in EGF distribution followed the administration of pilocarpine. It was concluded that EGF is secreted, at least partly into the saliva, following an alpha-adrenergic response, and that this occurs with degranulation of the cells of the CGT.     

Overexpression of epidermal growth factor induced hypospermatogenesis in transgenic mice

The in vivo role of epidermal growth factor (EGF) is not well defined even though its effects on culture cells were well studied. To understand the developmental, physiological, and pathological roles of EGF, we have generated transgenic mice widely expressing human EGF with the use of the beta-actin promoter. EGF and transforming growth factor alpha (TGFalpha) bind with equal affinity to the EGF receptor, a transmembrane tyrosine kinase, to trigger various biological responses. EGF and TGFalpha signaling are implicated in the development of the reproductive system. EGF also plays a physiological role in reproduction. Removal of the salivary gland in rodents, which reduces circulating EGF, reduces spermatogenesis, which can be corrected by EGF replacement. Here we show that in our transgenic males, only few post-meiosis II gametes were found, and the mice were sterile. This resembles a common cause of infertility in humans. Furthermore, the transgenic males had reduced serum testosterone. Our findings contrast the previous report on transgenic mice overexpressing TGFalpha in testis, which showed normal spermatogenesis. These data suggest that EGF is the active ligand for EGF receptor reported in germ cells, and proper EGF expression is important for completion of spermatogenesis.     

Localisation of epidermal growth factor receptor in mucous cells of human salivary glands

EGFR activation has been related to an increase in synthesis and secretion of mucins in epithelial cells, so that the use of EGFR tyrosine kinase inhibitors has been proposed in the therapy of mucin hypersecretory diseases. In this paper, we describe the ultrastructural localisation of EGFR in the mucous elements of human major and minor salivary glands and relate it to mucin distribution. A post-embedding immunogold staining method has been applied to normal surgical samples of human submandibular, sublingual, and labial glands, using a mouse monoclonal antibody specific for the intracellular domain of human EGFR. In mucous cells of all the glands examined, specific reactivity was detected in the cytoplasmic basolateral portions and near the mucous droplets, but not on cell surfaces. Since this pattern of labelling must be related to the internalisation process of the ligand-GFR complex, our results support the hypothesis that EGFR activation takes place in mucous cells and affects mucin production in human salivary glands.     

Basic fibroblast growth factor in rat salivary glands

We studied the occurrence and localization of basic fibroblast growth factor (bFGF) in rat salivary glands using a specific monoclonal antibody. It was shown that the extract of rat salivary glands has a pronounced stimulatory activity on the growth of bovine capillary endothelial cells, which is blocked by the addition of an antibody against bFGF. The concentration of bFGF in the submandibular/sublingual gland, as determined by radioimmunoassay, was 80% that in the brain. Immunocytochemistry revealed bFGF-immunoreactivity localized primarily in the epithelial cells lining the striated ducts and excretory ducts of the parotid, sublingual and submandibular glands. In addition, intense bFGF-immunoreactivity was observed in the granular convoluted tubule of the submandibular gland, localized predominantly in the agranular pillar cells, which lay in small numbers among the majority of weakly immunostained cells containing many apical secretory granules. At the electron-microscopic level, the immunoreactive material was distributed diffusely in the cytoplasmic matrix and nuclei of all immunoreactive cells, whereas it was absent from all cytoplasmic organelles including the secretory granules. These results indicate that bFGF is localized in different cellular and subcellular compartments from those of other growth factors in the duct system of rat salivary glands.     

Immunocytochemical localization of nerve growth factor in mouse salivary glands

Summary The submandibular glands of female mice and the sublingual and parotid glands of adult male and female mice have been examined by light microscopical immunocytochemistry for nerve growth factor (NGF). In female submandibular glands, staining for NGF was observed in granular convoluted tubule and striated duct cells. Sublingual glands of the mouse contained relatively few granular cells staining for NGF compared with submandibular glands. However, such granular cells appeared to be more numerous in male sublingual glands than in female glands. The remainder of the intralobular duct cells in both male and female sublingual glands exhibited apical subluminal staining for NGF as well as light basal plasmalemmal staining. Parotid glands in both male and female mice exhibited a similar pattern of staining for NGF in striated duct cells. However, the glands did not contain granular cells nor did they exhibit any pattern of staining which reflected a sexual dimorphism. Immunodot staining of salivary gland extracts confirmed the presence of immunoreactivity for NGF in all three of the major salivary glands.     

Expression of epidermal growth factor in salivary adenoid cystic carcinoma

This study used an immunohistochemical technique to assess the expression of epidermal growth factor (EGF) in 40 specimens of salivary adenoid cystic carcinoma (ACC), 7 specimens of labial glands adjacent to mucocele, and 5 specimens of normal submandibular glands. In normal submandibular glands, immunohistochemically detectable EGF was demonstrated in all ductal segments, including intercalated, striated, and excretory duct cells. No EGF positive staining was found in acinar compartments. including serous and mucous acinar cells. In degenerated labial glands adjacent to mucocele, no EGF staining was detected in the remaining acinar and ductal cells. In salivary ACCs, positive EGF immunostaining was observed in one of the 5 (20%) ACCs with a solid pattern and in 13 of the 35 (37.1%) ACCs with a tubular-cribriform pattern. The overall EGF expression rate in 40 salivary ACCs was 35%. Positive EGF staining was predominantly found in tubular structures in the tubular ACCs and in duct-like structures in large cribriform patterns or in the stroma of the cribriform ACCs. There was no significant correlation between EGF expression in salivary ACCs and any of the clinicopathological parameters including patient age and sex, cancer location, TNM status, clinical stage, histologic type, perivascular or perineural invasion, focal necrosis of tumor, and cellular atypia. We conclude that the duct segments of the normal submandibular gland are the sites of EGF synthesis and secretion. In degenerated labial glands adjacent to mucocele, EGF synthesis is completely inhibited. Furthermore, EGF is mainly biosynthesized in cells forming tubular or duct-like structures in tubular or cribriform salivary ACCs, and EGF may play a biologic role, particularly as a mitogen in salivary ACC growth.     

Relative growth of the skull and postcranium in giant transgenic mice

Cross-sectional allometric growth patterns of the cranial and postcranial skeleton were compared between giant transgenic (MT-rGH) mice and their normal littermate controls. Body weights, external body dimensions, and a series of cranial and postcranial linear dimensions of the skeleton were determined for samples of known age. Comparative bivariate and multivariate allometric analyses were completed in order to determine whether (1) the larger transgenic mice differed significantly from the normal controls in aspects of body and skeletal proportions, and (2) any such proportion differences resulted from general allometric effects of overall weight or skeletal size increase. Results demonstrate that the transgenic mice do exhibit significantly different body and skeletal proportions than normal control adults. Allometric comparisons of the skeletal dimensions relative to body weight reveal similar coefficients of growth allometry but several differences in gamma-intercept values in the transgenic vs. control groups. The comparisons among the skeletal dimensions of the skull and postcranium generally reveal the sharing and differential extension of common growth allometries in the two groups. Thus, the elevated levels of growth hormone (GH) and insulin-like growth factor I (IGF-I) in the transgenic mice appear to result in increased overall growth for the various skeletal elements, but in the relative proportions determined by intrinsic growth controls within that system.     

Induction of epidermal growth factor by tri-iodo-L-thyronine in the submandibular glands of mice with testicular feminization

The content of epidermal growth factor (EGF) as a high molecular weight complex (HMW-EGF) in the submandibular glands of mice was measured simply by a single radial immunodiffusion method. In female mice, the amount of HMW-EGF was increased 10-fold by tri-iodo-L-thyronine (T3) and 60-fold by 5 alpha-dihydrotestosterone (5 alpha-DHT). In mice with testicular feminization (Tfm), which are genetically deficient in androgen receptor, T3 but not 5 alpha-DHT increased the HMW-EGF from a non-detectable level to 5.4 +/- 0.94 micrograms/mg protein. It was concluded that EGF is also synthesized under the control of thyroid hormone in vivo, and that androgen was not involved in this induction of EGF by thyroid hormone.     

Stress-induced secretion of human growth hormone in transgenic mice

The effect of stress on human growth hormone (hGH) secretion was studied in transgenic mice. Experiments were conducted on fourth, fifth, and sixth generation male mice carrying a fusion gene, consisting of the promoter sequence of the mouse metallothionein I gene ligated to the hGH structural gene (mMT-I/hGH). In animals adapted to a controlled photoperiod, basal (unstimulated) levels of plasma hGH exhibited a diurnal cycling, with peak values occurring during the later half of the light period (15.5 +/- 1.0 vs 10.7 +/- 0.9 ng/ml, mean +/- SE, light versus dark, respectively). Food deprivation (5 days) led to elevated levels of plasma hGH (11.0 +/- 0.7 vs 32.0 +/- 4.2 ng/ml, preversus post-fast, respectively) accompanied by weight loss (49.5 +/- 0.8 vs 34.3 +/- 0.7 g), and hypoglycemia (7.8 +/- 0.2 vs 5.0 +/- 0.3 mM); glucose administration (5% drinking solution ad libitum) blocked the changes in levels of plasma hGH (12.2 +/- 1.1 vs 13.8 +/- 0.8 ng/ml) and plasma glucose (7.4 +/- 0.3 vs 7.9 +/- 0.5 mM), although the animals still sustained significant weight loss (44.9 +/- 1.6 vs 35.2 +/- 1.1 g). Vigorous exercise (swimming, 4 hr) produced a small but significant increase in plasma hGH, 12.1 +/- 1.1 ng/ml (1 hr pre-swim) vs 16.7 +/- 0.6 ng/ml (immediately post-swim). These findings indicate that the mMT-I/hGH transgene is responsive to the physiologic status of the host animal. Taken together with information regarding the heterologous components of the fusion gene, these data are consistent with the view that the hGH (structural) sequence may play a role in the response to stress.     

Increased activation of the epidermal growth factor receptor in transgenic mice overexpressing epigen causes peripheral neuropathy

In the mammalian nervous system, axons are commonly surrounded by myelin, a lipid-rich sheath that is essential for precise and rapid conduction of nerve impulses. In the peripheral nervous system (PNS), myelin sheaths are formed by Schwann cells which wrap around individual axons. While the tyrosine kinase receptors ERBB2 and ERBB3 are established mediators of peripheral myelination, less is known about the functions of the related epidermal growth factor receptor (EGFR) in the regulation of PNS myelination. Here, we report a peripheral neurodegenerative disease caused by increased EGFR activation. Specifically, we characterize a symmetric and distally pronounced, late-onset muscular atrophy in transgenic mice overexpressing the EGFR ligand epigen. Histological examination revealed a demyelinating neuropathy and axon degeneration, and molecular analysis of signaling pathways showed reduced protein kinase B (PKB, AKT) activation in the nerves of Epigen-tg mice, indicating that the muscular phenotype is secondary to PNS demyelination and axon degeneration. Crossing of Epigen-tg mice into an EGFR-deficient background revealed the pathology to be completely EGFR-dependent. This mouse line provides a new model for studying molecular events associated with early stages of peripheral neuropathies, an essential prerequisite for the development of successful therapeutic interventions.     

Biosynthetic human growth hormone: effects on growth of Snell dwarf mice

Bacterially synthesized human growth hormone (bhGH) administered to Snell dwarf mice during 4 weeks, induced an increase in body length and weight to a comparable degree as obtained with pituitary-derived human growth hormone (hGH). At a dose of 150 mU/day both bhGH and hGH induced a significant stimulation over saline-treated controls, of the weight of the submandibular salivary glands, the m. quadriceps femoris and gastrocnemius, the heart, liver, kidneys, thymus and spleen. The weight of the brain and the thickness of the skinfold were not influenced by either of the preparations used. When organ weights were expressed as a function of body weight, the contribution of the kidneys to body weight was significantly higher with hGH than with bhGH. The other organs studied did not show differences. As a biochemical parameter of cartilage growth, the sulfate incorporation into costal and epiphyseal cartilage in vitro was measured, and it was found to be stimulated by both hormones after short-term treatment. Thus bacterially synthesized hGH behaves identically to pituitary-derived hGH with respect to body length, sulfate incorporation into costal and epiphyseal cartilage, body weight and organ growth of Snell dwarf mice, with one exception: increase of weight of the kidneys, as a function of body weight, was more pronounced after treatment with hGH than with bhGH.     

Effects of the expression of bovine growth hormone on the testes and male accessory reproductive glands in transgenic mice

The effects of physiological and excessive levels of growth hormone (GH) on reproductive functions are poorly understood, and impairment of fertility is frequently observed in transgenic animals overexpressing GH genes. The present study was undertaken to determine the effects of chronic exposure to heterologous bovine GH (bGH) on the testes and accessory reproductive glands in transgenic mice. Endocrine function of the testes was evaluated by measuring the activities of two steroidogenic enzymes, 5-3-hydroxysteroid dehydrogenase (5-3-HSD) and 17-hydroxysteroid dehydrogenase (17-HSD). The activities of acid phosphatase, alkaline phosphatase and -glucuronidase, important hydrolytic enzymes of lysosomal origin, were measured in testes, seminal vesicles and ventral prostates in normal and transgenic mice. Testicular 5-3-HSD activity was higher in transgenic than in normal mice, while testicular 17-HSD activity in transgenic mice was not altered. Acid phosphatase activity was elevated in both seminal vesicles and ventral prostates of transgenic mice, while alkaline phosphatase activity was increased only in the prostate. The activity of -glucuronidase was elevated in the testes, seminal vesicles and ventral prostate gland of transgenic mice. These results suggest that chronic exposure to bGH is associated with significant stimulation of some hydrolytic enzymes in the testes and in the accessory reproductive glands of transgenic mice.     

Role of salivary glands and epidermal growth factor (EGF) in gastric secretion and mucosal integrity in rats exposed to stress

EGF, produced mainly by salivary glands, inhibits gastric acid secretion, stimulates the proliferation of gastric mucosal cells and protects the mucosa against various ulcerogens, but its role in the pathogenesis of stress ulcerations is unknown. In this study, rats with intact or resected salivary glands were exposed to water immersion and restraint stress (WRS) without and with pretreatment with exogenous EGF or dimethyl PGE2 (dmPGE2) at doses which were shown previously to protect the mucosa against topical irritants. During 1.5-12 h of WRS, the formation of gastric ulcerations increased progressively with the duration of stress reaching peak after 6 h of stress and being significantly higher in rats with removed salivary glands than in intact animals. Gastric acid secretion and DNA synthesis in oxyntic mucosa declined with the duration of WRS, but after sialoadenectomy a significant increase in gastric acid secretion and a further decline in DNA synthesis were observed after WRS. EGF contents in the gastric lumen and the gastric mucosa were several times higher in rats subjected to stress than in control unstressed animals, indicating that stress causes an extensive release of EGF. Both exogenous EGF (17 nmol/kg/h) and dmPGE2 (143 nmol/kg) prevented, in part, the formation of gastric lesions, while inhibiting gastric acid secretion both in rats with intact or resected salivary glands. We conclude that water immersion and restraint stress is accompanied by an excessive release of EGF, which appears to attenuate gastric secretion, enhances the DNA synthesis and may limit the formation of stress-induced gastric ulcerations.     

Sexual dimorphism and growth hormone regulation of a hybrid gene in transgenic mice.

The sexually dimorphic expression of the urinary protein genes of mice (Mup genes) in the liver is mediated by the different male and female temporal patterns of circulating GH. Normal females were induced to male levels when GH was administered by injection to mimic the male GH pattern, showing that expression at the male level does not require a male sex steroid status in addition to intermittent GH. Two Mup-alpha 2u-globulin hybrid transgenes with different Mup gene promoters showed sexually dimorphic expression, and their expression in females increased to male levels upon testosterone treatment. GH-deficient (lit/lit) mice did not express these transgenes, and GH-deficient females did not respond to testosterone treatment, showing that GH was required for induction. Both normal and GH-deficient females were induced to male levels when GH was administered by injection. This is the first report of a transgene responsive to GH. A transgene consisting of a Mup promoter fused to a Herpes simplex virus thymidine kinase reporter sequence also showed sexual dimorphism, although to a lesser degree. It was expressed at the same level in normal females and GH-deficient mice of both sexes and was induced when GH-deficient mice were treated with GH. We propose that this transgene has a basal constitutive expression, possibly due to the absence of any rodent DNA downstream of the promoter. Since expression of the transgene was significantly induced by GH, the GH response is due at least in part to sequences in the promoter region.