Anosmin-1 activates vascular endothelial growth factor receptor and its related signaling pathway for olfactory bulb angiogenesis.

Anosmin-1 is a secreted glycoprotein encoded by the ANOS1 gene, and its loss of function causes Kallmann syndrome (KS), which is characterized by anosmia and hypogonadism due to olfactory bulb (OB) dysfunction. However, the physiological function of anosmin-1 remains to be elucidated. In KS, disordered angiogenesis is observed in OB, resulting in its hypoplasia. In this study, we examined the involvement of anosmin-1 in angiogenic processes. Anosmin-1 was detected on the vessel-like structure in OB of chick embryos, and promoted the outgrowth of vascular sprouts as shown by assays of OB tissue culture. Cell migration, proliferation, and tube formation of endothelial cells were induced by treatment with anosmin-1 as well as vascular endothelial growth factor-A (VEGF-A), and further enhanced by treatment with both of them. We newly identified that anosmin-1 activated VEGF receptor-2 (VEGFR2) by binding directly to it, and its downstream signaling molecules, phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC). These results suggest that anosmin-1 plays a key role in the angiogenesis of developing OB through the VEGFR2-PLCγ1-PKC axis by enhancing the VEGF function.

Vascular Endothelial Growth Factor-A Exerts Diverse Cellular Effects via Small G Proteins, Rho and Rap.

Vascular endothelial growth factors (VEGFs) include five molecules (VEGF-A, -B, -C, -D, and placental growth factor), and have various roles that crucially regulate cellular functions in many kinds of cells and tissues. Intracellular signal transduction induced by VEGFs has been extensively studied and is usually initiated by their binding to two classes of transmembrane receptors: receptor tyrosine kinase VEGF receptors (VEGF receptor-1, -2 and -3) and neuropilins (NRP1 and NRP2). In addition to many established results reported by other research groups, we have previously identified small G proteins, especially Ras homologue gene (Rho) and Ras-related protein (Rap), as important mediators of VEGF-A-stimulated signaling in cancer cells as well as endothelial cells. This review article describes the VEGF-A-induced signaling pathways underlying diverse cellular functions, including cell proliferation, migration, and angiogenesis, and the involvement of Rho, Rap, and their related molecules in these pathways.

FGFR1 Analyses in Four Patients with Hypogonadotropic Hypogonadism with Split-Hand/Foot Malformation: Implications for the Promoter Region.

Heterozygous loss-of-function mutations of FGFR1 (fibroblast growth factor receptor 1) cause various disorders including hypogonadotropic hypogonadism with split-hand/foot malformation (HH-SHFM). We examined FGFR1 in four Japanese patients with HH-SHFM (cases 1-4) and the mother of case 4 with HH only. Cases 1 and 2 had heterozygous loss-of-function mutations with no dominant negative effect (c.289G>A, p.[G97S]; and c.2231G>C, p.[R744T]), and case 3 had a splice donor site mutation (c.1663+1G>T). Notably, case 4 had a maternally inherited 8,312 bp microdeletion that involved noncoding exon 1U and impaired FGFR1 expression. Furthermore, consistent with the presence of transcription-related histone marks (e.g., H3K4Me3, H3K4Me1, and H3K27Ac) and multiple transcription factor-binding sites around exon 1U, functional studies demonstrated a marked transactivation function of a 414-bp segment harboring the transcription start site. These results support the relevance of FGFR1 mutations to HH-SHFM, and argue for the presence of the FGFR1 core-promoter elements around exon 1U.

Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation.

Deregulated expression of fibroblast growth factor receptors (FGFRs) and their ligands plays critical roles in tumorigenesis. The gene expression of an alternatively spliced isoforms of FGFR3, FGFR3IIIc, was analyzed by RT-PCR in samples from patients with esophageal carcinoma (EC), including esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC). The incidence of FGFR3IIIc was higher in EC [12/16 (75%); p=0.073] than in non-cancerous mucosa (NCM) [6/16 (38%)]. Indeed, an immunohistochemical analysis of early-stage ESCC showed that carcinoma cells expressing FGFR3IIIc stained positively with SCC-112, a tumor marker, and Ki67, a cell proliferation marker, suggesting that the expression of FGFR3IIIc promotes cell proliferation. We used EC-GI-10 cells endogenously expressing FGFR3IIIc as a model of ESCC to provide mechanistic insight into the role of FGFR3IIIc in ESCC. The knockdown of endogenous FGFR3 using siRNA treatment significantly abrogated cell proliferation and the overexpression of FGFR3IIIc in cells with enhanced cell proliferation. EC-GI-10 cells and ESCC from patients with EC showed endogenous expression of FGF2, a specific ligand for FGFR3IIIc, suggesting that the upregulated expression of FGFR3IIIc may create autocrine FGF signaling in ESCC. Taken together, FGFR3IIIc may have the potential to be an early-stage tumor marker and a molecular target for ESCC therapy.

VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells.

Neuropilin-1 (NRP1) has been identified as a VEGF-A receptor. DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1. In the present study, the RNA interference of VEGF-A or NRP1 suppressed DJM-1 cell proliferation. Furthermore, the overexpression of the NRP1 wild type restored shNRP1-treated DJM-1 cell proliferation, whereas NRP1 cytoplasmic deletion mutants did not. A co-immunoprecipitation analysis revealed that VEGF-A induced interactions between NRP1 and GIPC1, a scaffold protein, and complex formation between GIPC1 and Syx, a RhoGEF. The knockdown of GIPC1 or Syx reduced active RhoA and DJM-1 cell proliferation without affecting the MAPK or Akt pathway. C3 exoenzyme or Y27632 inhibited the VEGF-A-induced proliferation of DJM-1 cells. Conversely, the overexpression of the constitutively active form of RhoA restored the proliferation of siVEGF-A-treated DJM-1 cells. Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor. A cell-penetrating oligopeptide that targeted GIPC1/Syx complex formation inhibited the VEGF-A-induced activation of RhoA and suppressed DJM-1 cell proliferation. In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.

A study on Borna disease virus infection in domestic cats in Japan.

Borna disease virus (BDV) infection causes neurological disease in cats. Here, we report BDV infection in 199 hospitalized domestic cats in the Tokyo area. BDV infection was evaluated by detection of plasma antibodies against BDV-p24 or -p40. BDV-specific antibodies were detected in 54 cats (27.1%). Interestingly, the percentage of seropositive cats was not significantly different among the three clinical groups, i.e., healthy (29.8%), neurologically asymptomatic disease (22.2%) and neurological disease (33.3%). The specific antibodies were present even in cats aged below one year. The seropositive ratio was constant, irrespective of age and sampling season. The present study suggests that additional factors are required for onset of Borna disease in naturally infected cats and that BDV is transmitted through vertical routes in cats.

Submicroscopic deletion involving the fibroblast growth factor receptor 1 gene in a patient with combined pituitary hormone deficiency.

Combined pituitary hormone deficiency (CPHD), isolated hypogonadotropic hypogonadism (IHH), Kallmann syndrome (KS), and septo-optic dysplasia (SOD) are genetically related conditions caused by abnormal development of the anterior midline in the forebrain. Although mutations in the fibroblast growth factor receptor 1 (FGFR1) gene have been implicated in the development of IHH, KS, and SOD, the relevance of FGFR1 abnormalities to CPHD remains to be elucidated. Here, we report a Japanese female patient with CPHD and FGFR1 haploinsufficiency. The patient was identified through copy-number analyses and direct sequencing of FGFR1 performed for 69 patients with CPHD. The patient presented with a combined deficiency of GH, LH and FSH, and multiple neurological abnormalities. In addition, normal TSH values along with a low free T4 level indicated the presence of central hypothyroidism. Molecular analyses identified a heterozygous ~ 8.5 Mb deletion involving 56 genes and pseudogenes. None of these genes except FGFR1 have been associated with brain development. No FGFR1 abnormalities were identified in the remaining 68 patients, although two patients carried nucleotide substitutions (p.V102I and p.S107L) that were assessed as benign polymorphism by in vitro functional assays. These results indicate a possible role of FGFR1 in anterior pituitary function and the rarity of FGFR1 abnormalities in patients with CPHD.

FGFR3 isoforms have distinct functions in the regulation of growth and cell morphology.

We have previously cloned the alternatively spliced isoform of fibroblast growth factor receptor 3 (FGFR3DeltaAB) that lacks the acid box in the extracellular region. To understand the biological functions and signal transduction of these FGFR3 isoforms, we analyzed the effect of FGF1 in ATDC5 cells, chondroprogenitor cell lines overexpressing these isoforms. In response to FGF1, FGFR3 induced a marked cell-morphology change to a round shape, while FGFR3DeltaAB did not. Furthermore, FGFR3 induced complete growth arrest, whereas FGFR3DeltaAB induced only moderate growth inhibition. Both receptors induced the expression of the CDK inhibitor p21(CIP1). However, only FGFR3 induced STAT1 phosphorylation that mediates the transcriptional induction of p21(CIP1), although both FGFR3 isoforms could induce a strong activation of mitogen-activated protein (MAP) kinases. Taken together, the different biological responses mediated by FGFR3 and FGFR3DeltaAB appear to be due to a difference in their ability to utilize STAT1 pathway and signals involved in cell rounding.