Specific 3-O-sulfated heparan sulfate domains regulate salivary gland basement membrane metabolism and epithelial differentiation [female]

FGF signaling is essential for salivary gland (SG) development and heparan sulfate (HS) regulation of FGFR function is determined by immense structural diversity of sulfated HS domains. The basement membrane (BM), containing HS proteoglycans, collagens and laminins, separates epithelia from stroma and controls growth factor-matrix cross-talk. 3-O-sulfotransferases generate highly 3-O-sulfated HS domains (3-O-HS) and Hs3st3a1 and Hs3st3b1 are enriched in myoepithelial cells (MECs), which produce BM and are a growth factor signaling hub. To investigate 3-O-HS regulation of MEC function and growth factor signaling, we generated Hs3st3a1;Hs3st3b1 double knockout (DKO) mice. The DKO HS loses specific highly 3-O-sulfated tetrasaccharides, which increases FGF/FGFR-complex binding to HS. During development this leads to increased FGFR-, BM- and MEC-related gene expression. However, in adult DKO SGs the secretory units containing acinar and MECs, have reduced MECs, increased BM and disrupted acinar polarity, resulting in salivary hypofunction. We used defined 3-O-sulfated-HS in FGFR pulldown assays and primary organ cultures to investigate 3-O-HS-dependent mechanisms regulating MEC development. We find that 3-O-HS modulates FGFR signaling to regulate MEC BM synthesis which is critical for secretory unit homeostasis and acinar function. Understanding how sulfated HS regulates development will inform the use of HS mimetics in organ regeneration. Overall design: Three submandibular glands from WT and DKO female mice were used to isolate RNA and perform RNA-sequencing to determine the differentially expressed genes.

成纤维细胞生长因子（Fibroblast Growth Factor, FGF）信号通路对唾液腺（Salivary Gland, SG）发育至关重要，而硫酸乙酰肝素（Heparan Sulfate, HS）对成纤维细胞生长因子受体（Fibroblast Growth Factor Receptor, FGFR）功能的调控，取决于硫酸化HS结构域的巨大结构多样性。基底膜（Basement Membrane, BM）含有蛋白聚糖、胶原蛋白与层粘连蛋白，可分隔上皮细胞与基质，并调控生长因子-基质串扰。3-O-硫酸转移酶（3-O-sulfotransferases）可生成高度3-O-硫酸化的HS结构域（3-O-HS），而Hs3st3a1与Hs3st3b1在肌上皮细胞（Myoepithelial Cells, MECs）中富集——这类细胞可合成基底膜，同时也是生长因子信号转导的枢纽。为探究3-O-HS对肌上皮细胞功能及生长因子信号转导的调控作用，我们构建了Hs3st3a1;Hs3st3b1双基因敲除（Double Knockout, DKO）小鼠。DKO小鼠的HS会丧失特异性的高度3-O-硫酸化四糖结构，这会增强FGF/FGFR复合物与HS的结合能力。在发育过程中，这会导致与FGFR、基底膜及肌上皮细胞相关的基因表达上调。然而在成年DKO小鼠的唾液腺中，包含腺泡细胞与肌上皮细胞的分泌单元出现了肌上皮细胞减少、基底膜增厚以及腺泡极性破坏的现象，最终引发唾液分泌功能减退。我们通过在FGFR下拉实验中使用定制的3-O-硫酸化HS，结合原代器官培养技术，探究了3-O-HS依赖的肌上皮细胞发育调控机制。研究发现，3-O-HS可通过调控FGFR信号通路，进而调节肌上皮细胞的基底膜合成——这一过程对分泌单元稳态及腺泡功能至关重要。阐明硫酸化HS的发育调控机制，将为硫酸乙酰肝素模拟物在器官再生中的应用提供理论依据。整体实验设计：选取野生型（Wild Type, WT）与DKO雌性小鼠各3个下颌下腺，提取RNA并开展RNA测序（RNA-sequencing），以鉴定差异表达基因（differentially expressed genes）。