pnas00316-0192 - Proc.Natl.Acad. Sci. USA Vol. 83,pp....

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Unformatted text preview: Proc.Natl.Acad. Sci. USA Vol. 83,pp. 4292-4296, June 1986 CellBiology Selectiveregulationoftrypsin gene expressionby calciumandby glucosestarvationinaratexocrinepancreascellline (AR4-2Jcelis/A23187/phorbol12-myristate13-acetate/acetyltransferaseassay) CHRISTIAN STRATOWA AND WILLIAM J.RUTTER Hormone ResearchInstituteandDepartmentsofBiochemistryandBiophysics,UniversityofCalifornia,San Francisco,CA 94143 Contributedby WilliamJ.Rutter,January2,1986 ABSTRACT Treatment ofthe ratpancreatic acinar cell line AR4-2J with the calcium ionophore A23187 selectively increases,withinafewhours,thesteady-stateleveloftrypsin mRNA. Addition of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate potentiates the calcium-in- duced increase.The mRNA leveloftheothertestedexocrine pancreaticgenesdecreases. These resultswere confrmed by DNA transfectionexperiments,usingthe5'flankingregionof the trypsin and chymotrypsin genes linked to the coding sequenceoftheacetyltransferase(CAT)gene. In calcium-induced cells transfected with the trypsin con- structs,anincreaseinCAT activitywasobserved,whereasthe chymotrypsinconstructs revealed a decreased CAT activity. GlucosestarvationofAR4-2Jcellssimilarlyelicitedaselective increaseintrypsinmRNA. Thisselectiveregulationoftrypsin may reflectitsroleasthekeyactivatoroftheotherzymogen species. Calcium isa majorintracellularmessenger stimulatingen- zymereleasefrompancreaticacinarcells(forreview,seeref. 1). It is unknown whether calcium is also involved in regulationoftheexpressionoftheexocrinepancreaticgenes. Could there be a correlation between Ca-induced enzyme secretionandCa-regulatedexpressionofthegenesforthose pancreatic enzymeswhose secretionistriggeredbycalcium? Early studiesrevealed a secretagogue-induced effluxof Ca2" inpancreaticfragments.More compellingevidencefor theimportanceof Ca2" insecretionwasprovidedbystudies inwhichanincreaseinintracellularfree Ca2+wasproduced by calcium ionophores, such as A23187 (1).The Ca iono- phoresstimulateamylase secretionfrompancreaticacini(2, 3).Furthermore,thetumor-promotingphorbolesterphorbol 12-myristate 13-acetate (PMA) has a synergistic effecton amylaserelease(4).SimilarexperimentsshowedthatA23187 and PMA in combination simulate the biphasicpattern of glucose-inducedinsulinsecretionfrompancreaticislets(5). These experimentssuggestthatthecalcium-activatedphos- pholipid-dependentproteinkinaseC(6)isalsoinvolvedinthe activationofsecretion. We have used theratpancreaticacinarcelllineAR4-2J, which displays a relatively high level of expression for severalexocrinegeneproducts(7),tostudytheinfluenceof calcium on the expression of these genes. Our studies revealed a selective induction oftrypsin by A23187 treat- ment, which is further potentiated by PMA. Using gene chimeras composed ofthe5'flankingregionsofthetrypsin orchymotrypsingeneslinkedtothecodingsequenceofthe bacterialacetyltransferase(CAT) gene(8), we demonstratedthatsequencesupstreamofthesegenesare...
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pnas00316-0192 - Proc.Natl.Acad. Sci. USA Vol. 83,pp....

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