RESUMO
The exquisite control of growth factor function by heparan sulfate (HS) is dictated by tremendous structural heterogeneity of sulfated modifications. How specific HS structures control growth factor-dependent progenitor expansion during organogenesis is unknown. We isolated KIT+ progenitors from fetal salivary glands during a stage of rapid progenitor expansion and profiled HS biosynthetic enzyme expression. Enzymes generating a specific type of 3-O-sulfated-HS (3-O-HS) are enriched, and fibroblast growth factor 10 (FGF10)/FGF receptor 2b (FGFR2b) signaling directly regulates their expression. Bioengineered 3-O-HS binds FGFR2b and stabilizes FGF10/FGFR2b complexes in a receptor- and growth factor-specific manner. Rapid autocrine feedback increases 3-O-HS, KIT, and progenitor expansion. Knockdown of multiple Hs3st isoforms limits fetal progenitor expansion but is rescued with bioengineered 3-O-HS, which also increases adult progenitor expansion. Altering specific 3-O-sulfated epitopes provides a mechanism to rapidly respond to FGFR2b signaling and control progenitor expansion. 3-O-HS may expand KIT+ progenitors in vitro for regenerative therapy.
Assuntos
Fator 10 de Crescimento de Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica , Heparitina Sulfato/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Células-Tronco/citologia , Sulfotransferases/metabolismo , Animais , Comunicação Autócrina , Western Blotting , Proliferação de Células , Feto/citologia , Feto/metabolismo , Fator 10 de Crescimento de Fibroblastos/genética , Imunofluorescência , Heparitina Sulfato/química , Imunoprecipitação , Hibridização In Situ , Camundongos , Técnicas de Cultura de Órgãos , Proteínas Proto-Oncogênicas c-kit/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândulas Salivares/citologia , Glândulas Salivares/metabolismo , Células-Tronco/metabolismo , Sulfotransferases/genéticaRESUMO
FGF10, a heparan sulfate (HS)-binding growth factor, is required for branching morphogenesis of mouse submandibular glands (SMGs). HS increases the affinity of FGF10 for FGFR2b, which forms an FGF10.FGFR2b.HS ternary signaling complex, and results in diverse biological outcomes, including proliferation and epithelial morphogenesis. Defining the HS structures involved in specific FGF10-mediated events is critical to understand how HS modulates growth factor signaling in specific developmental contexts. We used HS-deficient BaF3/FGFR2b cells, which require exogenous HS to proliferate, to investigate the HS requirements for FGF10-mediated proliferation and primary SMG epithelia to investigate the structural requirements of HS for FGF10-mediated epithelial morphogenesis. In BaF3/FGFR2b cells, heparin with at least 10 saccharides and 6-O-, 2-O-, and N-sulfates were required for maximal proliferation. During FGF10-mediated SMG epithelial morphogenesis, HS increased proliferation and end bud expansion. Defined heparin decasaccharide libraries showed that 2-O-sulfation with either an N-or 6-O-sulfate induced end bud expansion, whereas decasaccharides with 6-O-sulfation alone induced duct elongation. End bud expansion resulted from increased FGFR1b signaling, with increased FGFR1b, Fgf1, and Spry1 as well as increased Aqp5 expression, a marker of end bud differentiation. Duct elongation was associated with expression of Cp2L1, a marker of developing ducts. Collectively, these findings show that the size and sulfate patterns of HS modulate specific FGF10-mediated events, such as proliferation, duct elongation, end bud expansion, and differentiation, and provide mechanistic insight as to how the developmental localization of specific HS structures in tissues influences FGF10-mediated morphogenesis and differentiation.
