Pancreatic acinar cells: Molecular insight from studies of signal-transduction using transgenic animals

Pancreatic acinar cells are classical exocrine gland cells. The apical regions of clusters of coupled acinar cells collectively form a lumen which constitutes the blind end of a tube created by ductal cells – a structure reminiscent of a “bunch of grapes”. When activated by neural or hormonal secretagogues, pancreatic acinar cells are stimulated to secrete a variety of proteins. These proteins are predominately inactive digestive enzyme precursors called “zymogens”. Acinar cell secretion is absolutely dependent on secretagogue-induced increases in intracellular free Ca²⁺. The increase in [Ca²⁺]_i has precise temporal and spatial characteristics as a result of the exquisite regulation of the proteins responsible for Ca²⁺ release, Ca²⁺ influx and Ca²⁺ clearance in the acinar cell. This brief review discusses recent studies in which transgenic animal models have been utilized to define in molecular detail the components of the Ca²⁺ signaling machinery which contribute to these characteristics.     

Conditionally induced RAGE expression by proximal airway epithelial cells in transgenic mice causes lung inflammation

Background

Receptors for advanced glycation end-products (RAGE) are multiligand cell-surface receptors expressed abundantly by distal pulmonary epithelium. Our lab has discovered RAGE-mediated effects in the orchestration of lung inflammation induced by tobacco smoke and environmental pollutants; however, the specific contribution of RAGE to the progression of proximal airway inflammation is still inadequately characterized.

Methods and results

We generated a Tet-inducible transgenic mouse that conditionally overexpressed RAGE using the club cell (Clara) secretory protein (CCSP) promoter expressed by club (Clara) cells localized to the proximal airway. RAGE was induced for 40 days from weaning (20 days of age) until sacrifice date at 60 days. Immunohistochemistry, immunoblotting, and qPCR revealed significant RAGE up-regulation when compared to non-transgenic controls; however, H&E staining revealed no detectible morphological abnormalities and apoptosis was not enhanced during the 40 days of augmentation. Freshly procured bronchoalveolar lavage fluid (BALF) from CCSP-RAGE TG mice had significantly more total leukocytes and PMNs compared to age-matched control littermates. Furthermore, CCSP-RAGE TG mice expressed significantly more tumor necrosis factor alpha (TNF-α), interleukin 7 (IL-7), and interleukin 14 (IL-14) in whole lung homogenates compared to controls.

Conclusions

These data support the concept that RAGE up-regulation specifically in lung airways may function in the progression of proximal airway inflammation.     

Diabetes induced in male transgenic mice by expression of human H-ras oncoprotein in pancreatic beta cells.

Transgenic mice expressing an insulin-promoted H-ras hybrid gene in pancreatic beta cells developed beta-cell degeneration and diabetes. The disease was manifested in male mice by hyperglycemia, glycosuria, and reduced plasma insulin levels, which appeared around 5 months of age and led to premature death. Histological analyses revealed large holes within the islets of Langerhans and a reduced number of beta cells. The destruction of the islets was not associated with an obvious inflammatory activity. Ultrastructural analysis showed extensive engorgement in the endoplasmic reticulum of the residual beta cells from diabetic males. The females carrying the insulin-promoted ras gene did not manifest any of the physiological abnormalities observed in males and showed only minor histological and ultrastructural changes, even at much greater ages.     

Regional dysfunction correlates with myofiber disarray in transgenic mice with ventricular expression of ras

A hallmark of certain cardiac diseases such as familial hypertrophic cardiomyopathy is focal myofiber disarray. Regional ventricular dysfunction occurs in human subjects with hypertrophic cardiomyopathy; however, no direct evidence exists to correlate regional dysfunction with myofiber disarray. We used a transgenic mouse, which exhibits regional myofiber disarray via ventricular expression of the human oncogene ras, to investigate the relationship between myofiber disarray and septal surface strain. An isolated ejecting mouse heart preparation was used to record deformation of markers on the septal surface and to determine nonhomogeneous septal surface strain maps. Myofiber disarray made in histological tissue sections was correlated with gradients in surface systolic shortening. Significantly smaller maximum principal shortening was associated with disarray located near the right ventricle (RV) septal surface. There was also significantly smaller surface shear strain associated with disarray located either near the RV surface or at the midwall. Because surface shear is a local indicator of torsion, we conclude that myofiber disarray is associated with reduced septal torsion and reduced surface shortening.     

Signal transduction of MCP-1 expression induced by pancreatitis-associated ascitic fluid in pancreatic acinar cells

Pancreatitis-associated ascitic fluid (PAAF) is known to contribute to the progression of acute pancreatitis (AP). We have investigated the capability of PAAF to activate the expression of MCP-1 in pancreatic acinar cells and the involvement of MAPK, NF-κB and STAT3 as downstream signalling transduction pathways. The actions of dexamethasone (Dx) and N-acetylcysteine (NAC) on the PAAF's acinar effects have also been evaluated. Acinar cells were incubated for 1 hr with PAAF collected from rats with severe AP induced by sodium taurocholate in the absence or presence of Dx (10⁻⁷ M) or NAC (30 mM). MCP-1 mRNA expression, phospho-p38-MAPK, IκBα, nuclear p65 levels and nuclear translocation of STAT3 were analysed. In response to PAAF, overexpression of MCP-1, phosphorylation of p38-MAPK, degradation of IκBα and increases in p65 nuclear levels and STAT3 activity were found in acinar cells. PAAF-mediated MCP-1 up-regulation was completely suppressed by Dx and NAC. MAPK activation was only inhibited by NAC, NF-κB activation was repressed by Dx and NAC, and STAT3 pathway was strongly blocked by Dx and significantly reduced by NAC. In conclusion, acinar cells were activated by PAAF to produce MCP-1, mainly via NF-κB and STAT3 pathways. Both downstream pathways were targeted by Dx and NAC to repress the PAAF-mediated acinar MCP-1 up-regulation.     

Conditional apoptosis induced by oncogenic ras in thyroid cells

Mutations of ras are tumor-initiating events for many cell types, including thyrocytes. To explore early consequences after oncogenic Ras activation, we developed a doxycycline-inducible expression system in rat thyroid PCCL3 cells. Beginning 3-4 days after H-Ras(v12) expression, cells underwent apoptosis. The H-Ras(v12) effects on apoptosis were decreased by a mitogen-activated protein kinase kinase (MEK1) inhibitor and recapitulated by doxycycline-inducible expression of an activated MEK1 mutant (MEK1(S217E/S221E)). As reported elsewhere, acute expression of H-Ras(v12) also induces mitotic defects in PCCL3 cells through ERK (extracellular ligand-regulated kinase) activation, suggesting that apoptosis may be secondary to DNA damage. However, acute activation of SAPK/JNK (stress-activated protein kinase/Jun N-terminal kinase) through acute expression of Rac1(v12) also triggered apoptosis, without inducing large-scale genomic abnormalities. H-Ras(v12)-induced apoptosis was dependent on concomitant activation of cAMP by either TSH or forskolin, in a protein kinase A-independent manner. Thus, coactivation of cAMP-dependent pathways and ERK or JNK (either through H-Ras(v12), Rac1(v12), or MEK1(S217E/S221E)) is inconsistent with cell survival. The fate of thyrocytes within the first cell cycles after expression of oncogenic Ras is dependent on ambient TSH levels. If both cAMP and Ras signaling are simultaneously activated, most cells will die. Those that survive will eventually lose TSH responsiveness and/or inactivate the apoptotic cascade through secondary events, thus enabling clonal expansion.     

Pancreatic acinar cells utilize tyrosine to synthesize L-dihydroxyphenylalanine

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.     

Craniofacial abnormalities induced by ectopic expression of the homeobox gene Hox-1.1 in transgenic mice

Hox-1.1 is a murine homeobox-containing gene expressed in a time- and cell-specific manner during embryogenesis. We have generated transgenic mice that ectopically express Hox-1.1 from the chicken beta-actin promoter. In these mice Hox-1.1 expression was changed to an almost ubiquitous pattern. Ectopic expression of Hox-1.1 leads to death of the transgenic animals shortly after birth and is associated with multiple craniofacial anomalies, such as cleft palate, open eyes at birth, and nonfused pinnae. This phenotype is similar to the effects seen after systemic administration of retinoic acid during gestation. This suggests that retinoic acid embryopathy and the specific developmental defects caused by ectopic expression of a potential developmental control gene share a common pathogenic mechanism.     

Enhanced expression of Harvey ras induced by serum deprivation in cultured astrocytes

Trophic deprivation contributes to astrocyte damage that occurs in acute and chronic neurodegenerative disorders. Unraveling the underlying mechanisms may pave way to novel cytoprotective strategies. Cultured mouse astrocytes responded to trophic deprivation with a large and transient increase in the expression of p21^ras, which was secondary to an enhanced formation of reactive oxygen species (ROS) detected by cytofluorimetric analysis after preloading with 2',7'-dichlorofluorescein diacetate. The increase in p21^ras levels was largely attenuated by the reducing agent, N -acetylcysteine, which was proven to reduce ROS formation in astrocytes subjected to serum deprivation. We extended the analysis to the Ha-Ras isoform, which has been implicated in mechanisms of cytotoxicity. We found that serum deprivation enhanced the expression and activity of Ha-Ras without changing Ha-Ras mRNA levels. The increase in Ha-Ras levels was sensitive to the protein synthesis inhibitor, cycloheximide, suggesting that serum deprivation increases translation of preformed Ha-Ras mRNA. The late decline in Ha-Ras levels observed after 60 min was prevented by the proteasome inhibitor, MG132, as well as by the selective mitogen-activated protein kinase (MAPK) inhibitor, PD98059. Serum deprivation led to the activation of the MAPK pathway in cultured astrocytes, as shown by an increase in phosphorylated extracellular signal-regulated kinase 1/2 levels after 5 and 30 min. Finally, using the siRNA technology, we found that an acute knock-down of Ha-Ras was protective against astrocyte damage induced by serum deprivation. We conclude that cultured astrocytes respond to trophic deprivation with an increased expression in Ha-Ras, which is limited by the concomitant activation of the MAPK pathway, but is nevertheless involved in the pathophysiology of cell damage.     

Insulin-dependent diabetes mellitus induced in transgenic mice by ectopic expression of class II MHC and interferon-gamma

We have produced transgenic mouse strains harboring class II major histocompatibility complex or interferon-gamma genes linked to the human insulin promoter. These experiments were designed to investigate the consequences of the expression of immunological effector molecules by nonimmunological cells. In both of these studies we observed the disappearance from the pancreas of the insulin-producing beta cells coinciding with the development of insulin-dependent diabetes mellitus. Transgenic mice expressing both chains of the I-A gene showed progressive atrophy of the islets of Langerhans, whereas mice expressing interferon-gamma suffered an inflammatory destruction of the islets.     

Persistent ultrastructural changes in pancreatic acinar cells induced by 4-acetylaminofluorene

Male Leeds rats were fed a diet containing 0.05% of the non-carcinogen 4-acetylaminofluorene (4-AAF) for 8–10 months. They were then returned to a normal diet and their pancreatic tissues examined by electron microscopy at intervals between 2 and 12 months after the end of 4-AAF treatment. 4-AAF was found to induce a persistent alteration in the morphology of the granular endoplasmic reticulum, involving distortion and dilatation of the cisternae. In some respects this lesion resembles that which is induced by the carcinogenic isomer, 2-acetylaminofluorene (2-AAF).     

Chimeric prion protein expression in cultured cells and transgenic mice.

The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558-1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847-857; Prusiner, S.B., et al., 1990, Cell 63, 673-686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease-resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa-specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.     

Targeted expression of a conditional oncogene in hematopoietic cells of transgenic mice

We have produced two lines of transgenic mice in which the expression of temperature-sensitive SV-40 large T antigen is targeted to bone marrow megakaryocytes via the platelet factor 4 (PF4) tissue-specific promoter. The progeny of these transgenic mice were observed for about 3 mo, and no malignancies were detected over this period of time. The offspring of these transgenic mice, 6- to 12-wk of age, served as a source of bone marrow cells, which upon in vitro cultivation at the permissive temperature yielded immortalized cell lines (MegT). At the permissive temperature, MegT cells exhibit the characteristics of early 2N and 4N megakaryocytes which include the presence of specific gene products such as PF4, glycoprotein IIb, acetylcholinesterase, and CD45 as well as the absence of molecular markers of other cell lineages such as the macrophage marker Mac-1, the T helper cell marker CD4, the mast cell marker IgE, the T cell marker CD2 or the erythroid cell marker alpha-globin. The inactivation of the oncogene by a shift of temperature from 34 degrees to 39.5 degrees C produces a reduction in the frequency of the 2N cells, in conjunction with the appearance of 8N and 16N cells, consisting of 27 and 3% of total cells, respectively. Thus, we have generated hematopoietic cell lines that are trapped in the early stages of megakaryocyte commitment, but able to undergo part of the normal program of terminal differentiation.     

Development of mammary hyperplasia and neoplasia in MMTV-TGF alpha transgenic mice

To study the role of transforming growth factor alpha (TGF alpha) in normal mammary development and mammary neoplasia in vivo, we have generated transgenic mice in which a human TGF alpha cDNA is expressed under the control of the MMTV enhancer/promoter. Overexpression of TGF alpha in the mammary epithelium, as confirmed by in situ hybridization and immunohistochemistry, is associated with hyperplasia of alveoli and terminal ducts in virgin female and pregnant transgenic mice. A range of morphologic abnormalities including lobular hyperplasia, cystic hyperplasia, adenoma, and adenocarcinoma is seen in mammary tissue of transgenic females. In contrast, no morphologic abnormalities are seen in transgenic males in spite of TGF alpha overexpression in salivary glands and reproductive organs. TGF alpha can therefore act as an oncogene in vivo and appears to predispose mammary epithelium to neoplasia and carcinoma.     

Neurofilament gene expression in transgenic mice

1. DNA fragments that include the human neurofilament NF-L gene was found to be correctly expressed in the majority of neurons in transgenic mice. 2. The NF-L transgene product, which is detectable in situ with a species-specific monoclonal antibody, provides a powerful genotype marking system applicable to developmental and regeneration studies of the mammalian nervous system. 3. The proximal 5'-flanking region of the NF-L gene is sufficient to direct expression of a heterologous gene in the mouse nervous system.