Platelet-derived Growth Factor Receptor Regulates Salivary Gland Morphogenesis via Fibroblast Growth Factor Expression

Abstract

A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is β-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor α is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRα, and PDGFRβ were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme. A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is β-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor α is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRα, and PDGFRβ were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme. Platelet-derived growth factors (PDGFs), 2The abbreviations used are:PDGFplatelet-derived growth factorPDGFRPDGF receptorEnembryonic day nSMGsubmandibular glandNCneural crestBrdUrdbromo-2′-deoxyuridineERKextracellular signal-regulated kinaseMAPKmitogen-activated protein kinaseANOVAanalysis of variancesiRNAshort interfering RNAX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosidePBSphosphate-buffered salineECMextracellular matrixMMPmatrix metalloproteinaseRTreverse transcriptionMEKMAPK/ERK kinaseFGFfibroblast growth factorFGFRFGF receptor. originally identified as mitogenic factors for smooth muscle cells, fibroblasts, and glia cells, are also known as paracrine growth factors that mediate epithelial-mesenchymal interactions in various tissues (1Heldin C.H. Westermark B. Physiol. Rev. 1999; 79: 1283-1316Crossref PubMed Scopus (1996) Google Scholar). This growth factor family is known to have four members at present, PDGF-A, -B, -C, and -D, all of which assemble into homo- or heterodimer forms. PDGFs bind to two tyrosine kinase receptors, PDGFRα and -β, with PDGF-A, -B, and -C binding to PDGFRα as high affinity ligands and PDGF-B and -D binding to PDGFRβ (2Betsholtz C. Karlsson L. Lindahl P. BioEssays. 2001; 23: 494-507Crossref PubMed Scopus (297) Google Scholar). platelet-derived growth factor PDGF receptor embryonic day n submandibular gland neural crest bromo-2′-deoxyuridine extracellular signal-regulated kinase mitogen-activated protein kinase analysis of variance short interfering RNA 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside phosphate-buffered saline extracellular matrix matrix metalloproteinase reverse transcription MAPK/ERK kinase fibroblast growth factor FGF receptor. PDGFRα is expressed in the embryo by several different types of progenitor cells that proliferate and migrate in response to PDGF (3Lindahl P. Betsholtz C. Curr. Opin. Nephrol. Hypertens. 1998; 7: 21-26Crossref PubMed Scopus (46) Google Scholar). For example, PDGFRα is expressed in precursor cells that become cranial neural crest (NC) cells and is thought to be required for migration of those cells into the branchial arches. PDGFRα is also expressed by smooth muscle progenitors in developing lungs (4Lindahl P. Karlsson L. Hellstrom M. Gebre-Medhin S. Willetts K. Heath J.K. Betsholtz C. Development (Camb.). 1997; 124: 3943-3953PubMed Google Scholar) and widely throughout the embryonic mesenchyme (5Orr-Urtreger A. Lonai P. Development (Camb.). 1992; 115: 1045-1058PubMed Google Scholar). PDGFRα-deficient mice have a variety of defects in crest-derived tissues, including gross craniofacial, skeletal, and cardiac abnormalities (6Orr-Urtreger A. Bedford M.T. Do M.S. Eisenbach L. Lonai P. Development (Camb.). 1992; 115: 289-303PubMed Google Scholar, 7Morrison-Graham K. Schatteman G.C. Bork T. Bowen-Pope D.F. Weston J.A. Development (Camb.). 1992; 115: 133-142PubMed Google Scholar, 8Schatteman G.C. Morrison-Graham K. van Koppen A. Weston J.A. Bowen-Pope D.F. Development (Camb.). 1992; 115: 123-131PubMed Google Scholar, 9Payne J. Shibasaki F. Mercola M. Dev. Dyn. 1997; 209: 105-116Crossref PubMed Scopus (38) Google Scholar, 10Soriano P. Development (Camb.). 1997; 124: 2691-2700PubMed Google Scholar). These mice are deficient in functional cardiac NC cells, although it is unclear whether the deficiency is in the number of cells that reach target tissues, in NC cell differentiation, or functional target sites, such as cardiac and nasal septation defects and cleft face, cleft palate, and tooth defects (5Orr-Urtreger A. Lonai P. Development (Camb.). 1992; 115: 1045-1058PubMed Google Scholar, 10Soriano P. Development (Camb.). 1997; 124: 2691-2700PubMed Google Scholar, 11Ding H. Wu X. Kim I. Tam P.P. Koh G.Y. Nagy A. Mech. 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However, conditional mutant NCC-PDGFRα embryos do not exhibit defects in NC survival, migration, or proliferation, suggesting that PDGFRα is required for post-migratory NC functions (12Tallquist M.D. Soriano P. Development (Camb.). 2003; 130: 507-518Crossref PubMed Scopus (204) Google Scholar). Although PDGF-B and PDGFRβ knock-out mice die around the time of birth, the roles of PDGF-B in adult vascular, kidney, and retina pathogeneses have been elucidated in studies that used conditional knock-out mice, transgenic mice, and mice harboring mutations that alter PDGF activity without causing early lethality (16Boucher P. Gotthardt M. Li W.P. Anderson R.G. Herz J. Science. 2003; 300: 329-332Crossref PubMed Scopus (474) Google Scholar, 17Klinghoffer R.A. Mueting-Nelsen P.F. Faerman A. Shani M. Soriano P. Mol. Cell. 2001; 7: 343-354Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Kidneys and retinas are both particularly sensitive to mutations in the PDGFRβ signaling domain, as those affected exhibit the hallmarks of glomerulosclerosis and proliferative retinopathy (17Klinghoffer R.A. Mueting-Nelsen P.F. Faerman A. Shani M. Soriano P. Mol. Cell. 2001; 7: 343-354Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Together, these results indicate that PDGF-A and -B are critical for regulating organogenesis. The process of branching morphogenesis is utilized in all animals to form complex-ordered structures and involves dynamic and reciprocal interactions between different tissue the of Cell. 1999; 96: Full Text Full Text PDF PubMed Scopus Google Scholar, Science. 1999; PubMed Scopus Google Scholar). 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Development (Camb.). 2000; PubMed Google Scholar). embryos were for activity and for at in in phosphate-buffered saline embryos were in in were at a of and in in and in The embryos were and in were at a of and with mouse were and with in at The tissues were in for and into with a were with the which were in for with the used antibodies PDGF-A PDGF-B PDGFRα PDGFRβ and antibodies were by or antibodies A was used for and RNA was to the RNA was into in of of as a and of was in of of reverse transcription of and of the was in the The were time was with with by a was for at for for and for as H. S. T. Y. J. 2003; PubMed Scopus Google Scholar). organ cultures were in with and and with PDGF-AA or PDGF-BB for at the tissues were with of with with at for and which the were to a for the were by and analyzed by were or with The of was with and to and salivary gland to as in this were from mice and cultured at the The were of in The was with and were cultured at in of In of of exogenous of PDGF-AA or of PDGF-BB was to the The were and and the were at time was at of the were analyzed by to that a of the was The were in and at with results to with PDGF-AA or was to the for the were with for in and for in in the were with a of for at the were in and the with cells were a as K. S. A. K. T. H. M. A. K. Y. S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. T. B. T. A. Y. J. Biol. PubMed Scopus Google Scholar, A. Y. H. T. T. S. Biol. 2005; PubMed Scopus Google Scholar). was with for mouse PDGF-A and were with in to the M. K. K. A. T. K. Y. K. T. S. Biol. PubMed Scopus Google Scholar). of or for PDGF-A and PDGF-B was used for the SMGs. of PDGF and were cultured for as with neutralizing antibodies to PDGFs or antibodies to PDGF-AB and PDGF-BB were used for of and PDGF-BB as well as a and to at of of or of was of AG-17 and of were to the at the of FGF7 and were to the AG-17 were from were cultured with of the were cultured for and branching morphogenesis was as shown the between with were shown are and those with were to be from the of PDGFs in SMG branching we to the of NC cells in salivary glands the Wnt1-Cre/R26R mice, which is a transgenic mouse that results from with the conditional expression in all cells by expressed and it is for expression and cell the expression was to the of NC cells. cells were to mesenchymal cells in the SMG not in the epithelium This that embryonic SMG mesenchyme is derived from cranial NC cells. of PDGF-A, PDGF-B, and in we the of PDGF-A and -B, and PDGFRα and in the between epithelium and mesenchyme, we with antibodies for and which are of the PDGF-A was expressed in in the and of although it was in mesenchyme and PDGF-B and PDGFRα were expressed in mesenchymal cells in the and in epithelium, whereas PDGFRβ was in mesenchymal cells in the and not in the epithelium and are known to have epithelial and to branching morphogenesis. this PDGF-A and -B and PDGFRα and be found expressed in whereas for Pdgfa and is expressed all In this study, Pdgfb and expression was shown to be the A and the expression of PDGFs and their receptors in and mesenchymal tissue from SMG were by with and of and their receptors was analyzed by Pdgfa was expressed in epithelium and in mesenchyme, whereas that of and was in mesenchyme and not in epithelium, which was to and PDGF-AA and -BB and in SMG of and the we analyzed the of in SMG organ of was at with PDGF-AA and whereas that of was at and with PDGF-BB we epithelium and mesenchyme tissues from the SMG organ and stimulated with PDGF-AA or of was in SMG mesenchymal cells not in the epithelium These results that functional PDGFRα and are expressed in SMG mesenchyme and PDGF signaling into mesenchymal cells. we the functional of PDGFs organ the from mice showed in number of with the number as with However, the of PDGF-AA in branching morphogenesis in which was shown by in the number of by and as with the and results were with PDGF-BB and and in PDGF-AA and -BB gene expression were by analysis with PDGF-A and -B a of in the of PDGF-A and a in that of PDGF-B, as with the SMG cultures The of PDGF-A and -B inhibited branching morphogenesis at and with the of by and as A and and not have the branching morphogenesis of the not and the neutralizing for PDGF inhibited SMG branching morphogenesis. of SMG organ cultures with for PDGF-A and in the number of at and were with the at of SMG organ cultures with antibodies for PDGF-AB and in the of at and were with at analysis was by The for PDGF-AB to PDGF-AA and whereas that for PDGF-BB to of the to the SMG cultures inhibited branching morphogenesis at and and results were with the of the although the were as with and not have the branching morphogenesis of not Together, these results indicate that PDGF-A and PDGF-B are for the of SMGs. PDGF-AA and -BB and for PDGF-A and -B SMG the mechanism of branching morphogenesis stimulated by PDGF-AA and SMG was by by SMG epithelium the of PDGF-AA and -BB was increased by and as with the cultures with found to be increased in both the and of and In contrast, cells were not in the mesenchyme with of the and These results that PDGF-AA and -BB the of SMG epithelium and branching whereas do not have mesenchymal the of PDGF-A and -B branching we with SMG organ In the of the epithelial cells were whereas of mesenchymal cells were In cells, was a in the number of cells by as with the from PDGF-A and -B inhibited the of in the not in the cells and and and not have the of epithelium or mesenchymal cells not These indicate that the expression levels of PDGF-A and -B are branching morphogenesis of for the of epithelial cells. PDGF-AA and -BB of in and FGF by is expressed in SMG epithelium, although its receptor is expressed in mesenchyme. In the experiments, the of epithelial cells, not of mesenchyme, was with those we that factors from SMG mesenchyme regulate epithelial cell stimulated by was that are expressed in mesenchyme and branching morphogenesis in SMG cultures S. S. M. Development (Camb.). PubMed Scopus Google Scholar). we the expression of in SMG mesenchyme and organ cultures with PDGF-AA and -BB time PDGF-AA induced the expression of Fgf1, -3, -7, and -10, not of to PDGF-BB those and -10 were found to be expressed in SMG mesenchymal tissue and enhanced branching morphogenesis in studies S. S. M. Development (Camb.). PubMed Scopus Google Scholar, C. M. Development (Camb.). 2005; PubMed Scopus Google Scholar). In the experiments, PDGF-A and -B the expression of PDGF-A and -B and and inhibited the expression of and -10 and and not have the expression of in not These results indicate that the expression of and -10 in SMG mesenchymal cells be by PDGF-A and by PDGFs FGF inhibited PDGF receptor tyrosine kinase and decreased branching morphogenesis in SMG cultures is known to have the of the PDGF receptor expressed in mesenchyme and downstream In experiments, in SMG organ cultures stimulated by PDGF-BB was inhibited by the of and the inhibitor the of and -10 were inhibited in the of or However, FGF7 and were SMG branching morphogenesis that was inhibited by was fully indicating that function downstream of PDGF signaling A and and not the SMG branching morphogenesis that was inhibited by whereas of both FGF7 and -10 was of FGF7 or The known ligands for are -3, -7, and FGF7 and -10 bind with a high affinity to M. I. M. S. A. 2003; PubMed Scopus Google whereas to M. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the of PDGF induced SMG we and to morphogenesis by was inhibited in the of A and also inhibited SMG branching In of growth by was not by exogenous or although it was by of FGF7 and -10 not inhibited signaling and decreased branching morphogenesis by and was found to be expressed in the epithelial cells of SMGs. of the epithelial and not or become PDGF-BB not the inhibited branching morphogenesis of with and These that signaling is required for epithelial to and and indicate that PDGF function as of FGF the of SMG branching morphogenesis In this study, we analyzed the roles of PDGFs SMG epithelial PDGF-A and mesenchymal PDGF-B induced FGF expression in SMG mesenchyme and also enhanced branching morphogenesis. NC cells of a cell that is derived from the of the neural the early of Dev. Biol. 96: PubMed Scopus Google Scholar, J. Google Scholar). NC cells migrate as the branchial arches. these derived cells to the of mesenchymal structures in the and The a short and is expressed of the J.A. Cell. Full Text PDF PubMed Scopus Google Scholar, A. Cell. Full Text PDF PubMed Scopus Google Scholar). In a that used transgenic mouse of the the of NC cells that from the neural demonstrated for Y. Development (Camb.). Google Scholar). However, this transgenic cells be in which the of gene expression in the NC its activity the of NC cells. studies have also shown that of the with the conditional this and that NC cells in mice are Y. X. Y. Bringas Jr., P. J. Soriano P. Development (Camb.). 2000; PubMed Google Scholar, P. 1999; PubMed Scopus Google Scholar). In the mouse all cells in the mesenchyme become in the developing tooth Y. X. Y. Bringas Jr., P. J. Soriano P. Development (Camb.). 2000; PubMed Google and salivary glands PDGFRα is also a of cranial NC cells (4Lindahl P. Karlsson L. Hellstrom M. Gebre-Medhin S. Willetts K. Heath J.K. Betsholtz C. Development (Camb.). 1997; 124: 3943-3953PubMed Google Scholar, A. Bedford M.T. Do M.S. Eisenbach L. Lonai P. Development (Camb.). 1992; 115: 289-303PubMed Google the of this receptor or its ligands in salivary glands has not been mutant mice have a cleft and of the tooth X. Bringas Jr., P. Soriano P. Chai Y. Dev. Dyn. 2005; 232: 75-84Crossref PubMed Scopus (69) Google indicating that cells be critical for of the including the salivary In SMG organ PDGF-AA and -BB induced branching morphogenesis and also cell of and not of mesenchymal cells. and neutralizing antibodies for PDGFs to in SMG branching. These results that PDGFs regulate SMG branching morphogenesis. However, was not in SMG by or PDGFRα and PDGFRβ were expressed in SMG mesenchyme derived from NC cells. these we that mesenchymal factors induced by PDGFs regulate the of SMG epithelium and branching morphogenesis. In study, gene expression of showed that and were expressed in SMG epithelium S. S. M. Development (Camb.). PubMed Scopus Google whereas -3, and were expressed in mesenchyme. and transgenic mice do not salivary glands L. B. J. M. I. C. Development (Camb.). 2000; Google Scholar, S. J. 1998; PubMed Scopus Google Scholar). In signaling epithelial and branching morphogenesis to SMG organ These results that and are for epithelial and SMG branching morphogenesis. -3, -7, and -10 are expressed in mesenchyme, whereas Fgf1, and are expressed in both epithelium and mesenchyme S. S. M. Development (Camb.). PubMed Scopus Google Scholar). In this study, PDGF-AA and -BB induced the expression of Fgf1, -3, and -10 PDGF-A and -B a in Fgf1, -3, -7, and -10 in cultured indicating that PDGFs regulate the expression of in SMG mesenchymal cells. FGF7 and -10 and have positive the number and of SMG epithelial whereas and have as epithelial cell and the of cleft S. S. M. Development (Camb.). PubMed Scopus Google Scholar). In study, was decreased by PDGF-AA and -BB and increased by PDGF-A and -B which with the of of branching morphogenesis by and signaling regulates SMG epithelial morphogenesis suggesting that and increased of and as well as in both and are required for epithelium morphogenesis C. M. Development (Camb.). 2005; PubMed Scopus Google Scholar). is involved in and gland it epithelial not branching morphogenesis M. R.A. Dev. Biol. 2000; PubMed Scopus (72) Google Scholar, M. L. R.A. Development (Camb.). 2000; Google Scholar). In SMG epithelium, by at the of the C. M. Development (Camb.). 2005; PubMed Scopus Google Scholar). However, the of FGF7 and -10 in SMG mesenchyme are not In experiments, PDGF-AA and -BB increased SMG branching morphogenesis and FGF expression and and A and the signaling inhibitor AG-17 inhibited the of induced by with the of and -10 in salivary mesenchyme. AG-17 also inhibited SMG branching indicating that signaling in salivary mesenchyme is for FGF7 and -10 as well as SMG branching morphogenesis. In FGF7 the number of in the of the whereas PDGF-BB not the branching of in the of the inhibitor with and indicating that PDGF signaling of that of of the extracellular matrix and cell by matrix metalloproteinase and the family is a critical process in branching morphogenesis M. Y. M. A. Development (Camb.). 2001; Google Scholar). have including of such as and PDGF-BB the expression of in smooth muscle cells K. PubMed Scopus Google and PDGFs were found to the of and in cells C. A. M. J. Cell. Physiol. PubMed Scopus Google Scholar). These results that PDGFs regulate the expression of and of a mechanism for of at the of SMG epithelial In a study, a in expression by was shown to cleft whereas exogenous was not affected T. M. 2003; PubMed Scopus Google Scholar). is widely expressed in the and is also for organogenesis. embryos die the process of with including and and and salivary gland morphogenesis defects Li C. Dev. Biol. 2000; PubMed Scopus Google Scholar, S. H. K. H. Y. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Dev. Biol. PubMed Scopus (93) Google Scholar). decreased branching and expression of and in SMGs. for the expression of and exogenous branching morphogenesis of suggesting that signaling positive for has also been shown that mice have SMG branching, and embryos in SMG branching indicating that and by be for SMG branching morphogenesis M. T. Rev. Biol. 2000; PubMed Scopus Google Scholar, B. M. K. Dev. Biol. 2005; PubMed Scopus Google Scholar). of in salivary epithelium and mesenchyme that most are expressed in the mesenchyme, for and the two and C. M. Development (Camb.). 2005; PubMed Scopus Google Scholar). of around the of the and as well as of morphogenesis by MMP inhibitor in the epithelium in a study, that is for matrix and epithelial whereas and by be for SMG branching morphogenesis. roles for PDGF-A tissue morphogenesis are by the which a of the of the PDGF receptor. with this abnormalities K. Schatteman G.C. Bork T. Bowen-Pope D.F. Weston J.A. Development (Camb.). 1992; 115: 133-142PubMed Google and tissues from embryos exhibit in both and membrane-type MMP as with tissues from embryos (14Robbins J.R. McGuire P.G. Wehrle-Haller B. Rogers S.L. Dev. Biol. 1999; 212: 255-263Crossref PubMed Scopus (72) Google Scholar). In tooth of the gene not have the and of in the cranial neural crest-derived mesenchyme, although it the of the extracellular matrix and of cells in the in a growth X. Bringas Jr., P. Soriano P. Chai Y. Dev. Dyn. 2005; 232: 75-84Crossref PubMed Scopus (69) Google Scholar). the in in the mesenchyme of a critical for this extracellular in tooth Together, these that the expression of epithelial be by mesenchymal PDGF of the between PDGFs and salivary gland morphogenesis is in In we identified roles of PDGFs SMG branching morphogenesis. PDGF-AA and -BB enhanced SMG branching and induced Fgf1, -3, -7, and -10 in neural crest-derived mesenchymal tissues in SMGs. FGF expression and branching morphogenesis induced by PDGFs were inhibited by for PDGF-A and -B, as well as the inhibitor results indicate that epithelial branching induced by FGF be by PDGFs in a epithelial-mesenchymal P. for the for also for the with