Eleni Kousvelari

CELLULAR CHARACTERISTICS OF LONG-TERM CULTURED RAT PAROTID ACINAR CELLS

SummaryWe have successfully maintained and biochemically characterized differentiated rat parotid acinar cells cultured for long periods (6 mo.). The cells were cultured on a reconstituted basement membrane matrix in a medium containing a variety of agents that promote cellular proliferation and differentiation. The cultured cells retain the characteristics of the parental parotid acinar cells. They exhibit both secretory granules and abundant cellular organelles required for protein synthesis and secretion. In situ hybridization and immunocytochemistry demonstrate high levels of proline-rich protein mRNA and protein, and lower levels of amylase mRNA and protein, in their cytoplasm. These findings suggest that rat parotid acinar cells can be maintained in a differentiated state in vitro for long periods, and can serve as a useful model system for studying the regulation of exocrine secretory processes.

β-Adrenergic regulation of c-fos gene expression in an epithelial cell line

Stimulation of β‐adrenoreceptors in the RSMT‐A5 epithelial cell line is accompanied by an early and transient increase in the expression of the proto‐oncogene c‐fos. Maximal induction was at 30 min, returning to basal levels after 2 h. Similar results were obtained when cells were incubated with 8‐bromo‐cAMP. The induction of c‐fos is specific since the expression of p53, a transformation‐related gene, is not modulated by isoproterenol or 8‐bromo‐cAMP. The increase in c‐fos gene expression is not associated with proliferative activity in these epithelial cells.

Regulation of proto-oncogenes in rat parotid acinar cells in vitro after stimulation of β- adrenergic receptors

Stimulation of beta-adrenoreceptors in rat parotid acinar cells in vitro by the beta-adrenergic agonist isoproterenol induces steady-state levels of c-fos mRNA and c-fos protein in these cells. A dramatic increase in the steady-state levels of c-fos mRNA was observed at 60 min, followed by a decrease at 2 h with a second peak at 4 h. c-fos induction in rat parotid acinar cells in vitro seems to be mediated by cAMP. Increased levels of p53 and c-myc mRNA were detected only at 60 min. c-abl and c-sis were also induced by isoproterenol but in a pattern different from that seen with c-fos. c-abl was the only oncogene in rat parotid gland which showed increased expression after chronic isoproterenol treatment of rats. In rat parotid acinar cells we observed no correlation between DNA synthesis and c-fos induction.

Altered bacterial aggregation and adherence associated with changes in rat parotid-gland salivary proteins induced in vivo by β-adrenergic stimulation

Reduced adherence and aggregation were associated with protein alterations in parotid saliva after chronic treatment with the beta-adrenergic agonist isoproterenol. In contrast, saliva from animals treated with the beta-antagonist, propranolol, did not cause such changes; the protein composition of this saliva was similar to that of controls. SDS-polyacrylamide gel electrophoresis of protein in saliva samples before and after they were mixed with 10 mg of spheroidal hydroxyapatite beads (HA), as well as protein adsorbed and recovered from the HA, showed that an acidic, proline-rich protein with a molecular weight of approx. 40,000 was the predominant protein adsorbed. This protein was significantly diminished in saliva from isoproterenol-treated rats. Proteins with molecular weights between 44,000 and 48,000 and unique to the saliva from isoproterenol-treated animals were also adsorbed to HA. Thus alterations in proline-rich proteins of parotid saliva may influence the adherence and aggregation of oral bacteria, two processes considered important for in-vivo colonization of oral surfaces.

Regulation of salivary-gland-specific gene expression.

The results from in vivo transgenic and in vitro transfection studies designed to identify cis-element(s) and transfactor(s) governing the salivary proline-rich proteins (PRPs), amylase, and parotid secretory protein (PSP) gene expression are utilized as a paradigm to discuss the regulation of salivary-specific gene expression. Particular attention is given to the molecular mechanism(s) underlying the salivary PRP R15 gene regulation. In rodents, the PRPs are selectively expressed in the acinar cells of salivary glands, and are inducible by the beta-agonist isoproterenol and by dietary tannins. The results from a series of experiments using chimeric reporter constructs containing different lengths of the R15 distal enhancer region, their mutations, and various expressing constructs are analyzed and discussed. These data suggest that the inducible nuclear orphan receptor NGFI-B may participate in the regulation of salivary acinar-cell-specific and inducible expression of the rat R15 gene via three distinct distal NGFI-B sites. Taken together, a model for the induction of R15 gene expression by Ipr is proposed. However, the exact molecular basis of this NGFI-B-mediated transactivation of cAMP-regulated R15 expression remains to be established.

N-linked protein glycosylation in the rat parotid gland during aging

Abstract N-Linked protein glycosylation was examined in vitro in dispersed rat parotid acinar cells from young adult (3–6 months) and aged (22–24 months) rats. A small decrease in general protein production was observed with cells from aged animals (∼20% lower incorporation of [14C]leucine into 10% CCI3 COOH insoluble protein during continuous pulse labeling). Incorporation of [3H]mannose into N-linked glycoproteins by aged cells was further reduced (∼35%). Similarly microsomal membranes from parotid glands of aged animals showed ∼50% reduction in the synthesis of mannosylphosphoryl dolichol, a key intermediate in the dolichol pathway of protein N-glycosylation. Man-P-Dol synthase, the microsomal enzyme responsible for production of this saccharide-lipid, displayed no change in apparent K m for GDP-mannose when preparations from aged animals were utilized, but did show ∼50% reduction in V max. Following β-adrenoreceptor activation, cells from both young adult and aged glands showed increased N-linked protein glycosylation almost to the same extent (∼2-fold). The data suggested that in aged rat parotid cells there is a basal reduction of activity in the pathway responsible for asparagine-linked protein glycosylation, but that following exocytotic stimuli this pathway responds in a manner comparable to cells from young adult glands.

Characteristics of c-fos and jun B gene expression in A5 cells after β-adrenoreceptor stimulation and during the cell cycle

The beta-adrenoreceptor agonist isoproterenol elevates cAMP concentrations in the A5 rat salivary epithelial cell line and rapidly and transiently induces the expression of c-fos and jun B at 30 and 60 min following continuous stimulation of these cells. The induction of both genes is mediated by cAMP. We show here that the inducibility of these genes by isoproterenol or 8-BrcAMP is transcriptionally regulated and short (5 min) incubations of A5 cells with either agent is sufficient to trigger the induction of c-fos and jun B. We also have investigated the expression and inducibility of these genes during the A5 cell cycle. Both c-fos and jun B mRNA are elevated at the early phase of the cell cycle and are detectable throughout the cycle. At different stages of the cell cycle in synchronous A5 cells, both genes are as highly induced by isoproterenol or 8-BrcAMP as in asynchronous A5 cells. These studies provide the first evidence for the transcriptional regulation of c-fos and jun B by beta-adrenergic receptor stimulation or cAMP in an epithelial cell line (A5) and demonstrate the coordinate expression and inducibility of these genes at the different stages of the A5 cell cycle.

Dolichyl phosphate supplementation increases N-linked protein glycosylation in rat parotid acinar cells without increasing glycoprotein secretion.

N-linked protein glycosylation was increased three- to five-fold, in dispersed rat parotid acinar cells in vitro, by supplementation with exogenous dolichylphosphate. Despite this increase, glycoprotein secretion from both control and dolichylphosphate-supplemented cells was comparable.

Dissociation between c-fos gene expression and DNA synthesis in rat parotid glands.

Two experimental approaches were used to examine the relationship between c-fos gene expression and tissue proliferative responses. Beta-Adrenergic and muscarinic receptor stimulation yielded equivalent levels of c-fos expression, although only beta-adrenergic receptor agonists are reported as capable of eliciting DNA synthesis in parotid cells. Similarly, beta-adrenergic stimuli evoked comparable levels of c-fos expression in parotid cells from 2- and 12-month-old rats, whereas DNA synthesis has been shown to be much greater in younger animals. The results indicate that enhanced c-fos expression by itself is incapable of eliciting proliferative responses in rat parotid glands.

Influence of β-adrenergic stimulation on glycosylation of a major, secretory N-linked glycoprotein from rat parotid salivary gland

beta-Adrenergic stimulation with 10 microM isoproterenol increased the [3H]-mannose/[14C]-leucine ratio (three- to six-fold) of protein extracts in double-radiolabelled rat parotid acinar cells. Characteristics of oligosaccharides in a major parotid glycoprotein (Mr approximately 220,000; gp 220) were studied. Gp 220 from control and experimental cells was endoglycosidase H-insensitive, endoglycosidase F-sensitive and bound both concanavalin A and wheat germ agglutinin. Gp 220 was removed from concanavalin A-Sepharose by sequential elution with 10 mM alpha-methyl glucoside and 0.5 M alpha-methyl mannoside. These findings suggest that (1) oligosaccharides in gp 220 have both a biantennary complex and hybrid oligosaccharide chains, and (2) beta-adrenoreceptor stimulation has little effect on the gross oligosaccharide structures of this glycoprotein.