Phorbol 12-Myristate 13-Acetate-induced Ectodomain Shedding and Phosphorylation of the Human Meprinβ Metalloprotease

Abstract

Shedding of proteins localized at the cell surface is an important regulatory step in the function of many of these proteins. Human meprin (N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase, PPH, EC 3.4.24.18) a zinc-metalloendopeptidase of the astacin family is an oligomeric protein complex of α- and β-subunits and is expressed abundantly in the intestine and kidney as well as in leukocytes of the lamina propria and in cancer cells. In transfected cells intracellular proteolytic removal of the membrane anchor results in the secretion of the meprin α-subunit. In rats and mice, the β-subunit exists in a membrane-anchored form. In contrast, human meprinβ is constitutively converted into a secretable form. We now show that phorbol 12-myristate 13-acetate (PMA) stimulates an increased release of hmeprinβ from transfected COS-1 cells, whereas hmeprinα secretion is not influenced. This stimulatory effect is inhibited by the protein kinase C (PKC) inhibitor staurosporine, suggesting that activation of PKC mediates PMA-induced hmeprinβ shedding. The use of different protease inhibitors shows that two different metalloprotease activities are responsible for the constitutive and the PMA-stimulated hmeprinβ shedding. We identified tumor necrosis factor α-converting enzyme (TACE or ADAM17) as the protease that mediates the PMA-induced release. We also demonstrate that hmeprinβ is phosphorylated by PMA treatment on Ser687 within a PKC consensus sequence in the cytosolic domain of the protein. This phosphorylation of hmeprinβ is not, however, implicated in the enhanced secretion by PMA treatment. Shedding of proteins localized at the cell surface is an important regulatory step in the function of many of these proteins. Human meprin (N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase, PPH, EC 3.4.24.18) a zinc-metalloendopeptidase of the astacin family is an oligomeric protein complex of α- and β-subunits and is expressed abundantly in the intestine and kidney as well as in leukocytes of the lamina propria and in cancer cells. In transfected cells intracellular proteolytic removal of the membrane anchor results in the secretion of the meprin α-subunit. In rats and mice, the β-subunit exists in a membrane-anchored form. In contrast, human meprinβ is constitutively converted into a secretable form. We now show that phorbol 12-myristate 13-acetate (PMA) stimulates an increased release of hmeprinβ from transfected COS-1 cells, whereas hmeprinα secretion is not influenced. This stimulatory effect is inhibited by the protein kinase C (PKC) inhibitor staurosporine, suggesting that activation of PKC mediates PMA-induced hmeprinβ shedding. The use of different protease inhibitors shows that two different metalloprotease activities are responsible for the constitutive and the PMA-stimulated hmeprinβ shedding. We identified tumor necrosis factor α-converting enzyme (TACE or ADAM17) as the protease that mediates the PMA-induced release. We also demonstrate that hmeprinβ is phosphorylated by PMA treatment on Ser687 within a PKC consensus sequence in the cytosolic domain of the protein. This phosphorylation of hmeprinβ is not, however, implicated in the enhanced secretion by PMA treatment. Meprins, membrane-bound zinc-metalloendopeptidases of the astacin family and the metzincins superfamily were first identified in brush-border preparations of rodent and human kidney and intestinal epithelial cells (1Beynon R.J. Shannon J.D. Bond J.S. Biochem. J. 1981; 199: 591-598Crossref PubMed Scopus (101) Google Scholar, 2Sterchi E.E. Green J.R. Lentze M.J. Clin. Sci. 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Chem. 1994; 269: 15388-15393Abstract Full Text PDF PubMed Google Scholar). Meprin is capable of hydrolyzing many substrates in vitro, such as extracellular matrix proteins, hormones, and small peptides like gastrin 17, cerulein, and sCCK 8 of the gastrointestinal tract (6Sterchi E.E. Naim H.Y. Lentze M.J. Arch. Biochem. Biophys. 1988; 265: 119-127Crossref PubMed Scopus (34) Google Scholar, 9Kaushal G.P. Walker P.D. Shah S.V. J. Cell Biol. 1994; 126: 1319-1327Crossref PubMed Scopus (91) Google Scholar, 10Bertenshaw G.P. Turk B.E. Hubbard S.J. Matters G.L. Bylander J.E. Crisman J.M. Cantley L.C. Bond J.S. J. Biol. Chem. 2001; 276: 13248-13255Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Meprin may therefore be involved in the modulation of the activity of important cellular and extracellular proteins and peptides. We have observed elevated levels of meprin in patients with Crohn's disease 1D. Lottaz and E. E. Sterchi, unpublished data. and colon cancer (11Lottaz D. Maurer C.A. Hahn D. Büchler M.W. Sterchi E.E. Cancer Res. 1999; 59: 1127-1133PubMed Google Scholar). In addition we have found expression of meprin in leukocytes in the lamina propria of inflamed intestine, where it may be involved in cytokine processing (12Lottaz D. Hahn D. Müller S. Müller C. Sterchi E.E. Eur. J. Biochem. 1999; 259: 496-504Crossref PubMed Scopus (70) Google Scholar). Moreover a novel mRNA isoform (meprinβ′) was identified in a variety of human cancer cell lines like MCF7, SK-BR-3, U2OS, and BxPC3 (13Matters G.L. Bond J.S. Mol. Carcinog. 1999; 25: 169-178Crossref PubMed Scopus (39) Google Scholar). Studies of recombinant forms of meprin expressed in mammalian cells have yielded important information about the biosynthesis of both subunits (14Grünberg J. Dumermuth E. Eldering J.A. Sterchi E.E. FEBS Lett. 1993; 335: 376-379Crossref PubMed Scopus (36) Google Scholar, 15Milhiet P.E. Corbeil D. Simon V. Kenny A.J. Crine P. Boileau G. Biochem. J. 1994; 300: 37-43Crossref PubMed Scopus (14) Google Scholar, 16Johnson G.D. Hersh L.B. J. Biol. Chem. 1994; 269: 7682-7688Abstract Full Text PDF PubMed Google Scholar, 17Marchand P. Tang J. Johnson G.D. Bond J.S. J. Biol. Chem. 1995; 270: 5449-5456Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 18Eldering J.A. Grünberg J. Hahn D. Croes H.J. Fransen J.A. Sterchi E.E. Eur. J. Biochem. 1997; 247: 920-932Crossref PubMed Scopus (44) Google Scholar, 19Hahn D. Lottaz D. Sterchi E.E. Eur. J. Biochem. 1997; 247: 933-941Crossref PubMed Scopus (24) Google Scholar). In cells transfected with meprinα, cDNA proteolytic processing of the subunit occurs within the α-specific insertion domain. C-cytosolic and transmembrane domains are involved in retention and proteolytic processing of meprinα in the endoplasmic reticulum and are essential for subsequent intracellular transport (19Hahn D. Lottaz D. Sterchi E.E. Eur. J. Biochem. 1997; 247: 933-941Crossref PubMed Scopus (24) Google Scholar). The cleaved α-subunit is constitutively secreted into the medium when expressed alone and is largely retained at the cell surface in the presence of the β-subunit. Whereas mouse meprinβ is localized only at the cell surface (5Gorbea C.M. Marchand P. Jiang W. Copeland N.G. Gilbert D.J. Jenkins N.A. Bond J.S. J. Biol. Chem. 1993; 268: 21035-21043Abstract Full Text PDF PubMed Google Scholar, 8Marchand P. Tang J. Bond J.S. J. Biol. Chem. 1994; 269: 15388-15393Abstract Full Text PDF PubMed Google Scholar), human meprinβ is found at the cell surface and in a processed form in the culture medium of transfected cells and of cultured intestinal explants (12Lottaz D. Hahn D. Müller S. Müller C. Sterchi E.E. Eur. J. Biochem. 1999; 259: 496-504Crossref PubMed Scopus (70) Google Scholar, 18Eldering J.A. Grünberg J. Hahn D. Croes H.J. Fransen J.A. Sterchi E.E. Eur. J. Biochem. 1997; 247: 920-932Crossref PubMed Scopus (44) Google Scholar). We have recently shown that the soluble form of hmeprinβ is released from transfected cells by cleavage in front of the epidermal growth factor-like domain. A peptide sequence 13 amino acids long is necessary for this cleavage (20Pischitzis A. Hahn D. Leuenberger B. Sterchi E.E. Eur. J. Biochem. 1999; 261: 421-429Crossref PubMed Scopus (15) Google Scholar). The enzyme(s) involved and the regulation of this release are still unknown. Soluble forms of transmembrane proteins diverse in structure and function are generated by a process referred to as “ectodomain shedding.” The enzymes catalyzing this process are protein protein or and to the superfamily of that matrix a and COS-1 cells, kidney protein kinase phorbol 12-myristate tumor necrosis inhibitor of inhibitor of and and The release of membrane-bound proteins like J. 1999; PubMed Scopus Google Scholar, J.D. P. A.J. A.J. P. Sci. S. A. 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We also that PMA phosphorylation of hmeprinβ and we have identified Ser687 as the phosphorylation This phosphorylation step is not involved in the PMA-induced of however, the for a and meprin The of of hmeprinβ in to the constitutive secretion of hmeprinα from the cell surface different for the two meprin subunits in was from from Cell culture and were from and were from PMA and staurosporine, both from were in and as of and and were from was a from a from and a from and were from The protein were from were from for were by of phosphorylation in hmeprinβ for protein kinase Ser687 and were into and amino acids in mouse by the of recombinant A to and Scholar). The mammalian expression J.A. Grünberg J. Hahn D. Croes H.J. Fransen J.A. Sterchi E.E. Eur. J. Biochem. 1997; 247: 920-932Crossref PubMed Scopus (44) Google was as a were with the and In a these two were and the and the was with and and into the of The were by The and the were with the and and and cells transfected with hmeprinβ were with PMA for the of protease inhibitors on hmeprinβ shedding. of cells for in the presence of the the and cells were The cells were in in protease inhibitor and of the and cells were and to by to membrane The membrane was in by with hmeprinβ in for at in the membrane was with in The was to the to Cell and cells were in essential medium with and at in and cells were with 1987; PubMed Scopus Google Scholar). The of recombinant in of and of was to COS-1 cells. The cells were for at of the culture medium and subsequent for at The medium was with medium by a at were in medium for cells by were cultured in with and and were transfected with as by the of COS-1 and were with for at in and with of for at with was for the with medium medium with were and the medium was and In where PMA and/or the kinase inhibitor were cells were for in medium PMA PMA in the of cells, alone or with by the In cells addition of as with was the cells for at in medium addition of for in the presence of and was as J.A. Grünberg J. Hahn D. Croes H.J. Fransen J.A. Sterchi E.E. Eur. J. Biochem. 1997; 247: 920-932Crossref PubMed Scopus (44) Google Scholar, 19Hahn D. Lottaz D. Sterchi E.E. Eur. J. Biochem. 1997; 247: 933-941Crossref PubMed Scopus (24) Google Scholar). In cells, was in the presence of and to by in a to proteins were on and were as (19Hahn D. Lottaz D. Sterchi E.E. Eur. J. Biochem. 1997; 247: 933-941Crossref PubMed Scopus (24) Google Scholar). the were with as J.A. Grünberg J. Hahn D. Croes H.J. Fransen J.A. Sterchi E.E. Eur. J. Biochem. 1997; 247: 920-932Crossref PubMed Scopus (44) Google Scholar, 19Hahn D. Lottaz D. Sterchi E.E. Eur. J. Biochem. 1997; 247: 933-941Crossref PubMed Scopus (24) Google and for and treatment the proteins were in of The were in of and into were by treatment with of for at of and was by treatment with of of and of for at and were with from were with the from protein was by from cell and of Shedding by and secretion of hmeprinα and were in transfected COS-1 cells a The of hmeprinα of two with of and The the and the the cleaved form of hmeprinα as (14Grünberg J. Dumermuth E. Eldering J.A. Sterchi E.E. FEBS Lett. 1993; 335: 376-379Crossref PubMed Scopus (36) Google Scholar, 19Hahn D. Lottaz D. Sterchi E.E. Eur. J. Biochem. 1997; 247: 933-941Crossref PubMed Scopus (24) Google Scholar, E. Eldering J.A. Grünberg J. Jiang W. Sterchi E.E. FEBS Lett. 1993; 335: PubMed Scopus Google Scholar). A form was in medium of with PMA effect on hmeprinα secretion 8 and as also by of the of phorbol 12-myristate 13-acetate on secretion of human COS-1 cells, transfected with hmeprinα and or hmeprinβ and were with of for with or phorbol 12-myristate 13-acetate for and in culture medium for the proteins were to and by shown in A and C were and COS-1 cells in the presence and of phorbol 12-myristate 13-acetate The of medium forms was as from cell and the were and as of protein the of the medium forms were In COS-1 cells hmeprinβ the cell of two with of and with PMA in an enhanced secretion of the of hmeprinβ of and which was by a of protein. PMA-induced of hmeprinβ secretion was by In COS-1 cells of the hmeprinβ with the cellular membrane and only a was of the of hmeprinβ secreted into the medium was in cells with cells of in of the enzymes responsible for constitutive and PMA-induced of we have the of protease inhibitors on the of hmeprinβ in transfected COS-1 cells The of cleavage be in transfected cells hmeprinβ is expressed as a and therefore the metalloprotease inhibitors or not was also inhibited by the and inhibitors of and The inhibitors of and effect on hmeprinβ release The activity of the was in a not The of hmeprinβ by and was by an of the form of hmeprinβ results an of in the constitutive of The metalloprotease inhibitor P. S. Johnson R.J. 1997; PubMed Scopus Google also inhibited hmeprinβ release. a of in hmeprinβ cells were transfected with hmeprinβ transfected cells still secreted hmeprinβ this secretion was by PMA treatment This and the of hmeprinβ secretion in cells by that function is for PMA not for the constitutive hmeprinβ shedding. The constitutive is by a In to where effect on hmeprinβ the stimulatory effect of PMA in secretion of hmeprinβ was inhibited by A. A. P.E. C. V. A.J. G. FEBS Lett. PubMed Scopus Google Scholar). also shown to these the of in the PMA-induced of PMA the of of Human the of PMA treatment on proteolytic processing and secretion of the meprin in the presence or of PMA by subsequent with different were The two and forms were a of with and The was to treatment and to and that this was the form of The was to and a complex form of hmeprinβ that still with we observed with of and and and cleaved forms of hmeprinβ with with yielded two forms at and and The was observed PMA treatment of these cells. secreted hmeprinβ was in the culture medium and This secreted form not as by the to and the of cleaved and hmeprinβ in cell in the presence and of PMA only the A and and were cells in the of proteolytic processing of results that the intracellular of and cleaved protein is by PMA whereas the at the cell surface is enhanced phorbol and of PKC in the PMA-induced of Human of protein kinase in the PMA-induced of soluble hmeprinβ was the protein kinase inhibitor staurosporine, of the protein kinase C inhibitors Biochem. Biophys. Res. PubMed Scopus Google Scholar, J.S. Biochem. J. 1997; PubMed Scopus Google secretion of hmeprinβ at a and This secretion was enhanced by PMA and and the release was to levels of secreted protein by and that activation of a protein mediates PMA-induced hmeprinβ shedding. PMA of Human the of we the phosphorylation of hmeprinβ in transfected COS-1 cells. The domain of hmeprinβ PKC consensus with two PKC phosphorylation and COS-1 cells were transfected with and with in the presence of PMA by with a that both meprin subunits In COS-1 cells hmeprinα of hmeprinα a PKC phosphorylation of was observed In contrast, COS-1 cells transfected with the β-subunit of to a phosphorylated protein PMA treatment COS-1 cells transfected with the phosphorylation we identified Ser687 as the that the domain of hmeprinβ function as a PKC of of the or the phosphorylation of hmeprinβ is with the regulation of we the of PMA to secretion of hmeprinβ the phosphorylation of Ser687 and on the enhanced of hmeprinβ PMA A and This the that the release of human meprinβ is of phosphorylation and that occurs by an activity is by PMA the PKC PMA treatment of cells transfected with a where the transmembrane and cytosolic domains were by the from the α-subunit (19Hahn D. Lottaz D. Sterchi E.E. Eur. J. Biochem. 1997; 247: 933-941Crossref PubMed Scopus (24) Google Scholar), also in of the into the medium not This shows that the domain the phosphorylation is involved in the PMA of hmeprinβ We have that meprinα and meprinβ are secreted in a from transfected kidney cells (14Grünberg J. Dumermuth E. Eldering J.A. Sterchi E.E. FEBS Lett. 1993; 335: 376-379Crossref PubMed Scopus (36) Google Scholar, 18Eldering J.A. Grünberg J. Hahn D. Croes H.J. Fransen J.A. Sterchi E.E. Eur. J. Biochem. 1997; 247: 920-932Crossref PubMed Scopus (44) Google Scholar). of the meprinβ subunit was to the human as mouse meprinβ was not processing of hmeprinβ occurs and at the cell surface and a of 13 amino acids in the sequence (20Pischitzis A. Hahn D. Leuenberger B. Sterchi E.E. Eur. J. Biochem. 1999; 261: 421-429Crossref PubMed Scopus (15) Google Scholar). This is in to which is cleaved in the endoplasmic reticulum by a process that is on the α-specific insertion domain and the transmembrane P.E. Corbeil D. 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Full Text Full Text PDF PubMed Scopus Google Scholar, A.J. Biochem. J. 1997; PubMed Scopus Google Scholar, J. B. C. D. FEBS Lett. 1997; PubMed Scopus Google and are released treatment of cells with phorbol such as by protein kinase a cytosolic protein that membrane activation and intracellular proteins Biochem. J. 1993; PubMed Scopus Google Scholar). the of a kinase in the regulation of hmeprinβ were The effect of PMA-induced of hmeprinβ was by staurosporine, that protein kinase C is involved in the regulation of hmeprinβ shedding. Shedding to at or the cell The involved are have shown to important in the phorbol or release of membrane-anchored proteins P. S. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The that constitutive hmeprinβ an of in this This is by the with that an of hmeprinβ shedding. metalloprotease that is inhibited by is identified by to release the cytokine tumor necrosis factor P. S. Johnson R.J. 1997; PubMed Scopus Google Scholar). proteins like growth factor and are also by P. Johnson R.J. PubMed Scopus Google Scholar). This to the of cells to secretion of hmeprinβ was observed in these cells not by that constitutive and hmeprinβ are by different The protease responsible for the is in This is by the that an inhibitor of effect on the constitutive the PMA-stimulated of hmeprinβ in transfected COS-1 cells. have also by G. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google that of may be by PMA and that is the enzyme that mediates the PMA-induced not the constitutive release. The constitutive of hmeprinβ to be may be an or we have shown of hmeprinβ is also by B. Hahn D. A. Sterchi E.E. Biochem. J. PubMed Google Scholar). In to hmeprinα and mouse meprinα and hmeprinβ the of processing of hmeprinβ in transfected kidney cells was by an in secretion of the enzyme was suggesting that these have a proteolytic cleavage within the of at and were identified within the acid peptide found essential for hmeprinβ shedding. of COS-1 cells with a in which the 13 amino acids in a protein that was not and not the secreted of hmeprinβ was not these two be in the to the the meprinβ this acid peptide sequence is not B. Hahn D. A. Sterchi E.E. Biochem. J. PubMed Google Scholar), the cleavage to the release of the enzyme may be and be localized and Human in to two phosphorylation and within the protein kinase C phosphorylation in the cytosolic and J.A. Grünberg J. Hahn D. Croes H.J. Fransen J.A. Sterchi E.E. Eur. J. Biochem. 1997; 247: 920-932Crossref PubMed Scopus (44) Google Scholar, Mol. PubMed Scopus Google Scholar, B.E. Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar). This a of hmeprinβ in and/or regulation of proteolytic This phosphorylation is in human not in mouse a of in the function of this We have now phosphorylation of hmeprinβ in transfected COS-1 cells with This is the first phosphorylation of a of the astacin was identified as the of proteins on and is to in Cell Biol. 2001; PubMed Scopus Google Scholar, S.J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). A in hmeprinβ of phosphorylation may of an enzyme to the this the of secreted hmeprinβ in COS-1 cells, transfected with the were the the in a of the phosphorylated in the secretion of The of phosphorylation of hmeprinβ to for the regulation of by of a membrane-anchored protease P. J. Cell Sci. 2001; Google Scholar). is that phosphorylation the activity of The only activation for is the removal of the by or (14Grünberg J. Dumermuth E. Eldering J.A. Sterchi E.E. FEBS Lett. 1993; 335: 376-379Crossref PubMed Scopus (36) Google Scholar, S. Hahn D. Lottaz D. W. Sterchi E.E. J. 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J. 2001; PubMed Scopus Google Scholar). are to the function of the domain phosphorylation in meprinβ and membrane-bound such as The demonstrate that enhanced secretion as well as phosphorylation of hmeprinβ occurs PMA The of hmeprinβ in and/or disease is not In the small intestine, where both meprinα and are expressed in both subunits are on the brush-border membrane of these cells by (12Lottaz D. Hahn D. Müller S. Müller C. Sterchi E.E. Eur. J. Biochem. 1999; 259: 496-504Crossref PubMed Scopus (70) Google Scholar). The meprinβ subunit largely membrane-bound and the meprinα subunit is retained at the brush-border to the β-subunit by covalent is that of the proteolytic on the surface of is an for the of peptides and a in intestinal cells. In small intestinal both meprin subunits are also expressed in leukocytes of the lamina propria (12Lottaz D. Hahn D. Müller S. Müller C. Sterchi E.E. Eur. J. Biochem. 1999; 259: 496-504Crossref PubMed Scopus (70) Google Scholar). expression of soluble and membrane-bound in these cells may a of the proteolytic activity to the cell surface or to the extracellular on the of proteolytic activity be different Shedding of hmeprinβ may also be a of the proteolytic activity in a hmeprinα expression is to the gastrointestinal hmeprinβ a expression and E. E. Sterchi, unpublished data. and in have shown that a of different peptides and proteins, growth as well as extracellular matrix proteins are by (6Sterchi E.E. Naim H.Y. Lentze M.J. Arch. Biochem. Biophys. 1988; 265: 119-127Crossref PubMed Scopus (34) Google Scholar, 9Kaushal G.P. Walker P.D. Shah S.V. J. Cell Biol. 1994; 126: 1319-1327Crossref PubMed Scopus (91) Google Scholar, 10Bertenshaw G.P. Turk B.E. Hubbard S.J. Matters G.L. Bylander J.E. Crisman J.M. Cantley L.C. Bond J.S. J. Biol. Chem. 2001; 276: 13248-13255Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, D. W. Sterchi E.E. FEBS Lett. Scopus Google Scholar). is that substrates such as these are the for hmeprinβ in the In this it is of to an of and secreted in to substrates in a cell or may have that to a We E. for and were by and were a from and