Binding specificity between fibroblast growth factors (FGFs) and their receptors (FGFRs) is essential for mammalian development and is regulated primarily by two alternatively spliced exons, IIIb (''b'') and IIIc (''c''), that encode the second half of Ig-like domain 3 (D3) of FGFRs. FGF7 and FGF10 activate only the b isoform of FGFR2 (FGFR2b). Here, we report the crystal structure of the ligand-binding portion of FGFR2b bound to FGF10. Unique contacts between divergent regions in FGF10 and two b-specific loops in D3 reveal the structural basis by which alternative splicing provides FGF10-FGFR2b specificity. Structure-based mutagenesis of FGF10 confirms the importance of the observed contacts for FGF10 biological activity. Interestingly, FGF10 binding induces a previously unobserved rotation of receptor Ig domain 2 (D2) to introduce specific contacts with FGF10. Hence, both D2 and D3 of FGFR2b contribute to the exceptional specificity between FGF10 and FGFR2b. We propose that ligand-induced conformational change in FGFRs may also play an important role in determining specificity for other FGF-FGFR complexes. Binding specificity between FGFs and FGFRs is critical for the proper regulation of FGF signaling. The physiological significance of FGF-FGFR specificity is best demonstrated in Apert syndrome, a severe craniosynostosis syndrome, where point mutations in FGFR2 alter ligand binding affinity and specificity (8-10). Alternative splicing of FGFR mRNA is the main mechanism by which FGFRs with different ligand-binding profiles are generated. In FGFR1 to -3, D3 is encoded by the invariant exon IIIa followed by one of two alternative exons, IIIb (''b'') or IIIc (''c''). This alternative splicing event is regulated in a tissue-specific fashion with b expression restricted to epithelial lineages and c to mesenchymal lineages (11)(12)(13)(14). Most FGFs activate more than one FGFR. The FGF7 subfamily is unique among FGFs because its members (FGF7, FGF10, and FGF22) are expressed exclusively by mesenchyme and interact specifically with the b splice variant of FGFR2 (FGFR2b) resident in overlying epithelium (15-17).FGF7 and FGF10 bind FGFR2b with similar high affinity and compete with each other for this binding (18,19). However, striking phenotypic similarities between the FGF10 and FGFR2b knockout mice have established FGF10 as the predominant ligand for FGFR2b in developmental patterning and organogenesis. Both FGFR2b-null and FGF10-null mice die at birth and show agenesis of the lungs and limbs, whereas FGF7-null mice are viable and have normal lungs and limbs (16,(20)(21)(22).The exceptional specificity between the FGF7 subfamily and FGFR2b, together with the pivotal role of FGF10-FGFR2b signaling during development, makes the FGF10-FGFR2b complex an ideal model system for deciphering the structural determinants of FGF-FGFR binding specificity. In this report, we describe the crystal structure of the FGF10-FGFR2b complex and confirm the structural findings by mutational analysis. FGF10-FGFR2b specificity incorporate...

No abstract

No abstract

Receptor tyrosine kinases (RTKs) control a multitude of biological processes and are therefore subjected to multiple levels of regulation. Negative feedback is one of the mechanisms that provide an effective means to control RTK-mediated signaling. Sef has recently been identified as a specific antagonist of fibroblast growth factor (FGF) signaling in zebrafish and subsequently in mouse and human. Sef encodes a putative type I transmembrane protein that antagonizes the Ras͞mitogen-activated protein kinase pathway in all three species. Mouse Sef was also shown to inhibit the phosphatidylinositol 3-kinase pathway. We show here that an alternative splicing mechanism generates an isoform of human Sef, hSef-b, which unlike the previously reported Sef

Heparin-binding growth factors are crucial for the formation of human epidermis, but little is known about the role of heparan sulfate proteoglycans in this process. Here we investigated the role of the heparan sulfate proteoglycan, perlecan, in the formation of human epidermis, by utilizing in vitro engineered human skin. By disrupting perlecan expression either in the dermis or the epidermis, we found that epidermally derived perlecan is essential for epidermal formation. Perlecan-deficient keratinocytes formed a strikingly thin and poorly organized epidermis because of premature apoptosis and failure to complete their stratification program. Exogenous perlecan fully restored epidermal formation. Perlecan deposition in the basement membrane zone correlated with formation of multilayered epidermis. Perlecan deficiency, however, had no effect on the lining and deposition of major basement membrane components as was evident by a continuous linear staining of laminin and collagen IV. Similarly, perlecan deficiency did not affect the distribution of ␤1 integrin. Addition of the perlecan ligand, fibroblast growth factor 7, protected perlecan-deficient keratinocytes from cell death and improved the thickness of the epidermis. Taken together, our results revealed novel roles for perlecan in epidermal formation. Perlecan regulates both the survival and terminal differentiation steps of keratinocytes. Our results suggested a model whereby perlecan regulates these processes via controlling the bioavailability of perlecan-binding soluble factors involved in epidermal morphogenesis.Skin is the largest organ of the body. It serves as a shield against microorganism invasion and UV radiation, prevents dehydration, regulates body temperature, and is a part of the immune system (1). It consists of two distinct tissues, epidermis and dermis that are separated by a basement membrane (BM). 4 The dermis is a dense collagen-rich connective tissue that provides the support and nourishment to the overlying epidermis. It is mainly composed of fibroblasts that synthesize and secrete the various extracellular matrix (ECM) components (2). The epidermis is made primarily of keratinocytes that form a stratified squamous epithelium. It consists of multiple layers exhibiting distinct morphology and function. From the innermost to the outermost layers, they are the basal, spinous, granular, and cornified strata (3). Throughout adult life, the epidermis undergoes continuous self-renewal through proliferation of the basal cells, the only cells of the epidermis with the ability to proliferate. The keratinocytes undergo terminal differentiation as they leave the basal layer and move upward through the suprabasal layers toward the tissue surface, where they die and are sloughed off (3). This process makes the skin an excellent model system to study the coordinated regulation of cell proliferation, cell differentiation, and cell death. The formation of the mature epidermis is regulated by cross-talk with the adjacent connective tissue through a netw...

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.

IMPORTANCE Retinitis pigmentosa (RP) is the leading cause of incurable inherited blindness in the developed world, with an estimated prevalence of 1 in 3500 individuals. Therefore, it is important to develop new treatments for this disease. OBJECTIVE To determine the effect of oral treatment with 9-cis β-carotene on visual function of patients with RP. DESIGN Randomized, double-masked, placebo-controlled, crossover clinical trial. SETTING University tertiary medical facility. PARTICIPANTS Thirty-four patients with RP who were at least 18 years of age. Twenty-nine patients completed the study and were included in the analysis. INTERVENTIONS Patients were treated daily for 90 days with capsules containing 300 mg of 9-cis β-carotene-rich alga Dunaliella bardawil (β-carotene, approximately 20 mg) or placebo (starch). Following a 90-day washout period, they were treated for 90 days with the other capsules. MAIN OUTCOMES AND MEASURES The primary outcome was the change for both eyes from baseline to the end of each treatment in dark-adapted maximal electroretinographic b-wave amplitude. The secondary outcomes were the changes in light-adapted maximal b-wave amplitude, dark-and light-adapted visual field, and best-corrected visual acuity. RESULTS The mean change in dark-adapted maximal b-wave amplitude relative to initial baseline was +8.4 μV for 9-cis β-carotene vs −5.9 μV for placebo (P = .001). Ten participants (34.5%) had an increase of more than 10 μV for both eyes (range, 11-42 μV) after 9-cis β-carotene treatment compared with no participants after placebo treatment. The percentage change in light-adapted b-wave response was +17.8% for 9-cis β-carotene vs −3.0% for placebo (P = .01). No significant differences were found between the groups for visual field and best-corrected visual acuity. No adverse effects were observed. CONCLUSIONS AND RELEVANCE Treatment with 9-cis β-carotene significantly increased retinal function in patients with RP under the tested conditions. The optimal therapeutic regimen will be determined in future, larger clinical trials. 9-cis β-Carotene may represent a new therapeutic approach for some patients with RP. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01256697

Vision incapacitation and blindness associated with incurable retinal degeneration affect millions of people worldwide. In this study, 0.25×10(6) human bone marrow stem cells (hBM-MSCs) were transplanted epiretinally in the right eye of Royal College Surgeons (RCS) rats at the age of 28 days. Epiretinally transplanted cells were identified as a thin layer of cells along vitreous cavity, in close proximity to the retina or attached to the lens capsule, up to 6 weeks following transplantation. Epiretinal transplantation delayed photoreceptor degeneration and rescued retinal function up to 20 weeks following cell transplantation. Visual functions remained close to normal levels in epiretinal transplantation rats. No inflammation or any other adverse effects were observed in transplanted eyes. Our findings suggest that transplantation of hBM-MSCs as a thin epiretinal layer is effective for treatment of retinal degeneration in RCS rats, and that transplanting the cells in close proximity to the retina enhances hBM-MSC therapeutic effect compared with intravitreal injection.