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First published online 24 October 2007
doi: 10.1242/dev.011171

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Heparanase cleavage of perlecan heparan sulfate modulates FGF10 activity during ex vivo submandibular gland branching morphogenesis

¹ Matrix and Morphogenesis Unit, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Bethesda, MD, USA.
² Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program, Bethesda, MD, USA.
³ School of Biomedical Engineering, University of New South Wales, Sydney, Australia.
⁴ Department of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
⁵ Cancer and Vascular Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel.

View larger version (82K): [in this window] [in a new window]   Fig. 1. Heparanase is expressed in mouse SMGs throughout development, mainly in the mesenchyme, and colocalizes with perlecan in the epithelial basement membrane. (A) RT-PCR analysis of heparanase, Fgf10, and perlecan were compared at various developmental stages. Gene expression was normalized to 29S and is expressed relative to gene expression at E12. Data were obtained from triplicate experiments, repeated three times, and are mean±s.d. (B) Relative abundance of gene expression comparing E13 epithelium with mesenchyme. cDNA were prepared from E13 epithelium separated from mesenchyme. Gene expression was normalized to 29S. (C) Immunolocalization of heparanase and perlecan in E13 SMGs cultured for 36 hours. Heparanase (green) localizes in the mesenchyme and colocalizes with perlecan (red) in the basement membrane. The images are of a single 2 µm confocal section. Nuclei stained with SYBR-green (blue).

View larger version (74K): [in this window] [in a new window]   Fig. 2. Inhibition of heparanase function decreases branching morphogenesis of mouse SMGs. Laminaran sulfate (LMS), which inhibits heparanase activity, decreases branching morphogenesis (A), whereas the unsulfated laminarin control (LM) does not. In addition, a function-blocking anti-heparanase antiserum (B) decreases branching morphogenesis in a dose-dependent manner, whereas the control serum does not. E12 SMGs were cultured in the presence of unsulfated laminarin (LM; 10 or 50 µg/ml) or LMS (10 and 50 µg/ml), a heparanase function-blocking antiserum (Ab733; 1, 2, 4 µl/well), or normal rabbit serum (4 µl/well shown) for 48 hours. The number of buds was expressed as a ratio of the number of buds at 48 hours/number of buds at 1 hour (T48/T1). At least five SMGs/condition were used and the experiment repeated at least three times. ANOVA; *P<0.05; **P<0.01.

View larger version (90K): [in this window] [in a new window]   Fig. 3. Inhibition of heparanase function by Ab733 is specifically rescued by FGF10. SMGs were cultured for 48 hours with 1 µl of function-blocking Ab733 (IC50 number of end buds) and increasing doses (see Materials and methods for concentrations) of exogenous FGF1 (10 ng/ml), FGF2 (10 ng/ml), FGF7 (100 ng/ml), FGF10 (100 ng/ml) and HB-EGF (20 ng/ml) were added. The number of buds was expressed as a ratio of the number of buds at 48 hours/number of buds at 1 hour (T48/T1). At least five SMGs per condition were used and the experiment was repeated at least three times. ANOVA compared with the Ab733 alone, **P<0.01.

View larger version (98K): [in this window] [in a new window]   Fig. 4. Recombinant heparanase increases branching morphogenesis of the intact SMG, increases phosphorylation of ERK1/2, and when added to isolated epithelium cultured in a 3D ECM, increases lateral branching, end bud clefting and duct elongation. (A) E12 SMGs were cultured with 5 µg/ml of either inactive (I), active (A), or unprocessed (U) forms of heparanase for 48 hours. (B) The number of buds was expressed as a ratio of the number of buds at 48 hours/number of buds at 1 hour (T48/T1). (C) Western blot analysis of phospho-ERK1/2 and total ERK1/2 after 48 hours of treatment with either inactive, active, or unprocessed heparanase resulted in an 3-fold increase in pERK1/2 with active and an 2.3-fold increase with unprocessed heparanase. (D) Isolated SMG epithelia were cultured with 200 ng/ml of FGF10 (a sub-optimal dose for growth) and treated with 5 µg/ml of either inactive (which appeared similar to a carrier control, not shown), active or unprocessed heparanase and compared after 48 hours with epithelia cultured with 500 ng/ml of FGF10. The total number of end buds was counted from at least five glands/condition and the experiments repeated twice. ANOVA compared with inactive heparanase; *P<0.05; **P<0.01.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Heparanase releases an FGF10-FGFR2b complex bound to ECM in a solid-phase assay, and pretreatment of the ECM by heparanase decreases FGF10-FGFR2b but not FGF1-FGFR2b binding. (A) FGF10-FGFR2b or FGFR2b alone, was incubated in a 96-well plate precoated with a laminin-111 ECM that contains 2% perlecan by ELISA (data not shown). Either inactive (control) or active heparanase was added to release the bound complex, and FGFR2b was detected by ELISA. Pretreatment of the ECM with heparanase resulted in a larger decrease in binding of both the complex and the receptor alone. (B) The binding of FGF10-FGFR2b was decreased by both heparanase and heparitinase treatment; however, FGF1-FGFR2b could still bind the remaining HS after heparanase but not heparitinase treatment. ECM-coated wells were incubated with or without inactive heparanase (control), active heparanase, bacterial heparitinase, or chondroitinase for 1 hour, followed by incubation with FGF10-FGFR2b or FGF1-FGFR2b (1 nM each) for 1 hour. ELISA assays were performed in triplicate and repeated at least three times.

View larger version (17K): [in this window] [in a new window]   Fig. 6. The FGF10-FGFR2b complex shows greater binding to purified intact HUAEC perlecan than FGF10 or FGFR2b alone, and binding of the complex is reduced by heparanase treatment. (A) SPR analysis of FGF10, FGFR2b, and FGF10-FGFR2b complex binding to intact perlecan. Proteins were diluted in HBS-P containing 0.1 µg/ml heparin. (B) SPR analysis of FGF10-FGFR2b binding to perlecan before and after heparanase treatment. Heparanase (5 µg/ml at 5 µl/minute) was applied to the chip surface. RU, response units.

View larger version (48K): [in this window] [in a new window]   Fig. 7. FGFR2b and the FGF10-FGFR2b complex colocalize with endogenous SMG perlecan HS, and the binding is decreased by heparanase treatment. A whole-mount ligand and carbohydrate engagement (LACE) assay using E13 SMGs shows that FGFR2b (A) and the FGF10-FGFR2b complex (B) colocalize with perlecan in the basement membrane. Increased binding was detected with the complex compared to the receptor alone. There was decreased binding of both FGFR2b and the FGF10-FGFR2b complex after heparanase or heparitinase treatment. In addition, the FGF10-FGFR2b complex also colocalizes with syndecan 1 in the epithelium (C), but also binds other epithelial HSPGs, and the staining was not decreased by pretreatment with either heparanase or heparitinase (data not shown). Images are single confocal sections. Scale bar: 20 µm in A and B; 10 µm in C.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Model shows release of FGF10-bound HS fragments from perlecan by heparanase in the basement membrane. HS fragments modulate the biological activity of FGF10 by increasing FGF10-FGFR2b complex formation to promote MAPK phosphorylation, end bud growth and clefting, and lateral branch formation. Syndecan 1 in the epithelium binds FGF10-FGFR2b, and we speculate (grey text) that other unidentified epithelial HSPG may specify the location of lateral buds or end bud clefting. Epithelial HSPGs may form a signaling complex with FGFR2b, in combination with HS fragments released by heparanase, and increase MAPK or other intracellular signaling.