An Isolated Laminar Osteoma Arising in the Maxillary Sinus.

BACKGROUND Osteomas are benign tumors characterized by proliferation of dense or trabecular bone. Most osteomas of the head and neck occur in the mandible, they rarely occur in the maxillary sinus, and free osteomas are extremely rare. While usually detected incidentally on plain radiographs, symptoms appear when the osteoma obstructs the sinus orifice or invades the adjacent orbit or intracranial structures. Herein, we report a case of a patient with an isolated laminar osteoma arising in the maxillary sinus. CASE REPORT A 52-year-old man presented to our hospital with a radiopaque mass in the right maxillary sinus. An oval mass of size 2.7×2.3 cm was observed in the right maxillary sinus on computed tomography, and no sinusitis was present. Under general anesthesia, the mass was removed orally via the modified Caldwell-Luc method. Histopathologic examination revealed layered compact bony tissue covered by the sinus membrane. The mass was free from the inferior wall of the right maxillary sinus, and a part of the mass was covered by the sinus membrane. The postoperative course was uneventful, and no recurrence was noted after 5 years. CONCLUSIONS We experienced a case of an extremely rare osteoma in the maxillary sinus. The osteoma was solitary and free within the maxillary sinus. The treatment performed was surgical excision using the modified Caldwell-Luc method. The cause of the free osteoma was thought to be odontogenic maxillary sinusitis caused by apical periodontitis of the nearby tooth.

A Case of a Dentigerous Cyst in the Maxillary Sinus Treated Preoperatively With Vascular Embolization to Avoid Intraoperative Abnormal Bleeding.

Bone cysts involving the maxillary sinus are frequently observed, and controlling bleeding from the maxillary or posterior superior alveolar arteries is extremely difficult when the surgical site extends into the palatine fossa or the wing socket behind the maxillary sinus. In this report, we describe a case wherein preoperative endovascular arterial embolization prevented bleeding owing to an unexpected vascular injury that occurred during the removal of a dentigerous cyst from the maxillary sinus. This resulted in a safe operation with less intraoperative bleeding. Although this approach carries the risk of complications, such as paralysis, around the affected area, the likelihood of such complications is low. This approach is useful for performing a safe surgery with minimal blood loss because it avoids the need for emergency hemostasis for major intraoperative hemorrhage.

miR-1260b inhibits periodontal bone loss by targeting ATF6ß mediated regulation of ER stress.

The expression profiles of exosomal microRNAs (miRNAs) are regulated by the microenvironment, and appropriate priming with mesenchymal stem cells (MSCs) is one of the strategies to enhance the paracrine potency of MSCs. Our previous work demonstrated that exosomes from tumor necrosis factor (TNF)-α-primed human gingiva-derived MSCs (GMSCs) could be a therapeutic tool against periodontitis, and that TNFα-inducible exosomal miR-1260b is essential for the inhibition of alveolar bone loss. However, the precise molecular mechanism underlying miR-1260b-mediated inhibition of osteoclastogenesis is not yet fully understood. Here, we found that the activating transcription factor (ATF)-6ß, a novel miR-1260b-targeting gene, is critical for the regulation of osteoclastogenesis under endoplasmic reticulum (ER) stress. An experimental periodontal mouse model demonstrated that induction of ER stress was accompanied by enhanced ATF6ß expression, and local administration of miR-1260b and ATF6ß siRNA using polyethylenimine nanoparticles (PEI-NPs) significantly suppressed the periodontal bone resorption. In periodontal ligament (PDL) cells, the ER stress inducer, tunicamycin, enhanced the expression of the receptor activator of NF-κB ligand (RANKL), while miR-1260b-mediated downregulation of ATF6ß caused RANKL inhibition. Furthermore, the secretome from miR-1260b/ATF6ß-axis-activated PDL cells inhibited osteoclastogenesis in human CD14+ peripheral blood-derived monocytes. These results indicate that the miR-1260b/ATF6ß axis mediates the regulation of ER stress, which may be used as a novel therapeutic strategy to treat periodontal disease.

Extracellular vesicles derived from GMSCs stimulated with TNF-α and IFN-α promote M2 macrophage polarization via enhanced CD73 and CD5L expression.

Immunoregulatory properties of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are promising. Gingival tissue-derived MSCs (GMSCs) have unique immunoregulatory capacity and secrete large amounts of EVs. Recent findings suggest that priming MSCs with inflammatory stimuli is an effective strategy for cell-free therapy. However, the precise mechanism by which the contents of EVs are customized has not been fully elucidated. Here, we show that EVs derived from GMSCs primed with a combination of two pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interferon-α (IFN-α), synergistically promote anti-inflammatory M2 macrophage polarization by increasing the expression of cluster of differentiation 73 (CD73) and CD5 molecule-like (CD5L). Expression of CD73 by TNF-α/IFN-α stimulation was transcriptionally upregulated by the activation of mammalian target of rapamycin signaling and nuclear translocation of hypoxia-inducible factor 1α in GMSCs. TNF-α/IFN-α treatment also significantly increased the expression of CD5L mRNA via the transcription factor DNA-binding protein inhibitor ID3 and liver X receptor. Interestingly, exosomal CD5L is a prerequisite for the synergistic effect of EVs-mediated M2 macrophage polarization. These results indicate that combined pre-licensing with TNF-α and IFN-α in GMSCs is ideal for enhancing the anti-inflammatory function of EVs, which contributes to the establishment of a therapeutic tool.

Combined application of geranylgeranylacetone and amelogenin promotes angiogenesis and wound healing in human periodontal ligament cells.

Amelogenin directly binds to glucose-regulated protein 78 (Grp78). Cell migration activity is expected to increase when human periodontal ligament cells (hPDLCs) overexpressing Grp78 are treated with amelogenin. Geranylgeranylacetone (GGA) is a drug that induces the expression of heat shock protein and is routinely used to treat gastric ulcers. Here, we investigated the changes in the properties and behavior of hPDLCs in response to treatment with GGA and the synergistic effects of amelogenin stimulation in hPDLCs pretreated with GGA for the establishment of a novel periodontal tissue regenerative therapy. We observed that GGA treatment increased Grp78 protein expression in hPDLCs and enhanced cell migration. Microarray analysis demonstrated that increased Grp78 expression triggered the production of angiopoietin-like 4 and amphiregulin, which are involved in the enhancement of angiogenesis and subsequent wound healing via the activation of hypoxia-inducible factor 1α and peroxisome proliferator-activated receptors as well as the phosphorylation of cAMP response element-binding protein and protein kinase A. Moreover, the addition of recombinant murine amelogenin (rM180) further accelerated hPDLC migration and tube formation of human umbilical vein endothelial cells due to the upregulation of interleukin-8 (IL-8), monocyte chemotactic protein 1, and IL-6, which are also known as angiogenesis-inducing factors. These findings suggest that the application of GGA to gingival tissue and alveolar bone damaged by periodontal disease would facilitate the wound healing process by inducing periodontal ligament cells to migrate to the root surface and release cytokines involved in tissue repair. Additionally, supplementation with amelogenin synergistically enhanced the migratory capacity of these cells while actively promoting angiogenesis. Therefore, the combined application of GGA and amelogenin may establish a suitable environment for periodontal wound healing and further drive the development of novel therapeutics for periodontal tissue regeneration.

Solitary Neurofibroma of the Hard Palate: A Case Report and Literature Review.

BACKGROUND Neurofibromas are benign tumors of neurological origin caused by the proliferation of Schwann cells and fibroblasts; they often occur in the skin and nerves as a symptom of von Recklinghausen disease. Solitary neurofibromas are also known to occur on their own, but solitary development in the hard palate is extremely rare and difficult to distinguish from schwannomas. The neural origin of solitary neurofibromas is also difficult to determine intraoperatively, and there have been no reports that clearly identify the neural origin of neurofibromas in the hard palate. CASE REPORT We report a case of a solitary neurofibroma originating in the hard palate in a 24-year-old woman. She presented to our department with a 1.2×0.8-cm dome-shaped left palate mass. After identification of the nerve at the source, the tumor was resected under general anesthesia. Histopathology was positive for S-100 and CD34 immunostaining, as well as for Alcian blue. Eventually, the mass was diagnosed as a neurofibroma. CONCLUSIONS Solitary neurofibromas originating in the hard palate are difficult to differentiate from other neoplastic lesions, especially schwannomas, based on clinical findings alone. Therefore, it is important to perform a biopsy and immunostaining of the biopsied specimens for S-100 and CD34. In neurofibromas, tumor cells are loose and delicate, often with wavy or serpentine nuclei, and S-100 protein-positive cells are sparser than in schwannomas. An overall pathological diagnosis should be made with regard to CD34, taking into account that schwannomas are CD34-negative and neurofibromas are CD34-positive.

Exosomes from TNF-α-treated human gingiva-derived MSCs enhance M2 macrophage polarization and inhibit periodontal bone loss.

Mesenchymal stem cell (MSC)-derived exosome plays a central role in the cell-free therapeutics involving MSCs and the contents can be customized under disease-associated microenvironments. However, optimal MSC-preconditioning to enhance its therapeutic potential is largely unknown. Here, we show that preconditioning of gingival tissue-derived MSCs (GMSCs) with tumor necrosis factor-alpha (TNF-α) is ideal for the treatment of periodontitis. TNF-α stimulation not only increased the amount of exosome secreted from GMSCs, but also enhanced the exosomal expression of CD73, thereby inducing anti-inflammatory M2 macrophage polarization. The effect of GMSC-derived exosomes on inflammatory bone loss were examined by ligature-induced periodontitis model in mice. Local injection of GMSC-derived exosomes significantly reduced periodontal bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and these effects were further enhanced by preconditioning of GMSCs with TNF-α. Thus, GMSC-derived exosomes also exhibited anti-osteoclastogenic activity. Receptor activator of NF-κB ligand (RANKL) expression was regulated by Wnt5a in periodontal ligament cells (PDLCs), and exosomal miR-1260b was found to target Wnt5a-mediated RANKL pathway and inhibit its osteoclastogenic activity. These results indicate that significant ability of the TNF-α-preconditioned GMSC-derived exosomes to regulate inflammation and osteoclastogenesis paves the way for establishment of a therapeutic approach for periodontitis.

Amelogenin Downregulates Interferon Gamma-Induced Major Histocompatibility Complex Class II Expression Through Suppression of Euchromatin Formation in the Class II Transactivator Promoter IV Region in Macrophages.

Enamel matrix derivatives (EMDs)-based periodontal tissue regenerative therapy is known to promote healing with minimal inflammatory response after periodontal surgery, i. e., it promotes wound healing with reduced pain and swelling. It has also been reported that macrophages stimulated with amelogenin, a major component of EMD, produce various anti-inflammatory cytokines and growth factors. We previously found that stimulation of monocytes with murine recombinant M180 (rM180) amelogenin suppresses major histocompatibility complex class II (MHC II) gene expression using microarray analysis. However, the detailed molecular mechanisms for this process remain unclear. In the present study, we demonstrated that rM180 amelogenin selectively downmodulates the interferon gamma (IFNγ)-induced cell surface expression of MHC II molecules in macrophages and this mechanism mediated by rM180 appeared to be widely conserved across species. Furthermore, rM180 accumulated in the nucleus of macrophages at 15 min after stimulation and inhibited the protein expression of class II transactivator (CIITA) which controls the transcription of MHC II by IFNγ. In addition, reduced MHC II expression on macrophages pretreated with rM180 impaired the expression of T cell activation markers CD25 and CD69, T cell proliferation ability, and IL-2 production by allogenic CD4+ T lymphocytes in mixed lymphocyte reaction assay. The chromatin immunoprecipitation assay showed that IFNγ stimulation increased the acetylation of histone H3 lysine 27, which is important for conversion to euchromatin, as well as the trimethylation of histone H3 lysine 4 levels in the CIITA promoter IV (p-IV) region, but both were suppressed in the group stimulated with IFNγ after rM180 treatment. In conclusion, the present study shows that amelogenin suppresses MHC II expression by altering chromatin structure and inhibiting CIITA p-IV transcription activity, and attenuates subsequent T cell activation. Clinically observed acceleration of wound healing after periodontal surgery by amelogenin may be partially mediated by the mechanism elucidated in this study. In addition, the use of recombinant amelogenin is safe because it is biologically derived protein. Therefore, amelogenin may also be used in future as an immunosuppressant with minimal side effects for organ transplantation or MHC II-linked autoimmune diseases such as type I diabetes, multiple sclerosis, and rheumatoid arthritis, among others.

Sprouty2 is involved in the control of osteoblast proliferation and differentiation through the FGF and BMP signaling pathways.

Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) play essential roles in bone formation and osteoblast activity through the extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad pathways. Sprouty family members are intracellular inhibitors of the FGF signaling pathway, and four orthologs of Sprouty have been identified in mammals. In vivo analyses have revealed that Sprouty2 is associated with bone formation. However, the mechanism by which the Sprouty family controls bone formation has not been clarified. In this study, we investigated the involvement of Sprouty2 in osteoblast proliferation and differentiation. We examined Sprouty2 expression in MC3T3-E1 cells, and found that high levels of Sprouty2 expression were induced by basic FGF stimulation. Overexpression of Sprouty2 in MC3T3-E1 cells resulted in suppressed proliferation compared with control cells. Sprouty2 negatively regulated the phosphorylation of ERK1/2 after basic FGF stimulation, and of Smad1/5/8 after BMP stimulation. Furthermore, Sprouty2 suppressed the expression of osterix, alkaline phosphatase, and osteocalcin mRNA, which are markers of osteoblast differentiation. Additionally, Sprouty2 inhibited osteoblast matrix mineralization. These results suggest that Sprouty2 is involved in the control of osteoblast proliferation and differentiation by downregulating the FGF-ERK1/2 and BMP-Smad pathways, and suppresses the induction of markers of osteoblast differentiation.

Amelogenin induces M2 macrophage polarisation via PGE2/cAMP signalling pathway.

OBJECTIVES: Amelogenin, the major component of the enamel matrix derivative (EMD), has been suggested as a bioactive candidate for periodontal regeneration. Apart from producing a regenerative effect on periodontal tissues, amelogenin has also been reported to have an anti-inflammatory effect. However, the precise molecular mechanisms underlying these effects remain unclear. In the present study, we examined the immunomodulatory effects of amelogenin on macrophages. DESIGN: Human phorbol 12-myristate 13-acetate (PMA)-differentiated U937 macrophages and CD14+ peripheral blood-derived monocytes (PBMC)-derived macrophages were stimulated with recombinant amelogenin (rM180). After performing a detailed microarray analysis, the effects of rM180 on macrophage phenotype and signal transduction pathways were evaluated by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, confocal microscopy and flow cytometry. RESULTS: The microarray analysis demonstrated that rM180 increased the expression of anti-inflammatory genes in lipopolysaccharide (LPS)-challenged macrophages after 24h, while it temporarily up-regulated inflammatory responses at 4h. rM180 significantly enhanced the expression of M2 macrophage markers (CD163 and CD206). rM180-induced M2 macrophage polarisation was associated with morphological changes as well as vascular endothelial growth factor (VEGF) production. rM180 enhanced prostaglandin E2 (PGE2) expression, and the activation of the cAMP/cAMP-responsive element binding (CREB) signaling pathway was involved in amelogenin-induced M2 macrophage polarisation. Blocking of PGE2 signaling by indomethacin specifically abrogated rM180 with or without LPS-induced M2 shift in PBMC-derived macrophages. CONCLUSION: Amelogenin could reprogram macrophages into the anti-inflammatory M2 phenotype. It could therefore contribute to the early resolution of inflammation in periodontal lesions and provide a suitable environment for remodeling-periodontal tissues.

Inhibition of Sprouty2 polarizes macrophages toward an M2 phenotype by stimulation with interferon γ and Porphyromonas gingivalis lipopolysaccharide.

Periodontitis is a chronic inflammatory disorder caused by specific bacteria residing in the biofilm, particularly Porphyromonas gingivalis (Pg). Sprouty2 (Spry2) functions as a negative regulator of the fibroblast growth factor (FGF) signaling pathway. We previously demonstrated that sequestration of Spry2 induced proliferation and osteogenesis in osteoblastic cells by basic FGF (bFGF) and epidermal growth factor (EGF) stimulation in vitro, but diminished cell proliferation in gingival epithelial cells. In addition, Spry2 knockdown in combination with bFGF and EGF stimulation increases periodontal ligament cell proliferation and migration accompanied by prevention of osteoblastic differentiation. In this study, we investigated the mechanisms through which Spry2 depletion by interferon (IFN) γ and Pg lipopolysaccharide (LPS) stimulation affected the physiology of macrophages in vitro. Transfection of macrophages with Spry2 small-interfering RNA (siRNA) promoted the expression of genes characteristic of M2 alternative activated macrophages, induced interleukin (IL)-10 expression, and enhanced arginase activity, even in cells stimulated with IFNγ and Pg LPS. In addition, we found that phosphoinositide 3-kinase (PI3K) and AKT activation by Spry2 downregulation enhanced efferocytosis of apoptotic cells by increasing Rac1 activation and decreasing nuclear factor kappa B (NFκB) p65 phosphorylation but not signal transducer and activator of transcription 1 (STAT1) phosphorylation. Collectively, our results suggested that topical administration of Spry2 inhibitors may efficiently resolve inflammation in periodontal disease as macrophage-based anti-inflammatory immunotherapy and may create a suitable environment for periodontal wound healing. These in vitro findings provide a molecular basis for new therapeutic approaches in periodontal tissue regeneration.

Grp78 Is Critical for Amelogenin-Induced Cell Migration in a Multipotent Clonal Human Periodontal Ligament Cell Line.

Periodontal ligament stem cells (PDLSCs) are known to play a pivotal role in regenerating the periodontium. Amelogenin, which belongs to a family of extracellular matrix (ECM) proteins, is a potential bioactive molecule for periodontal regenerative therapy. However, its downstream target molecules and/or signaling patterns are still unknown. Our recent proteomic study identified glucose-regulated protein 78 (Grp78) as a new amelogenin-binding protein. In this study, we demonstrate, for the first time, the cellular responses induced by the biological interaction between amelogenin and Grp78 in the human undifferentiated PDL cell line 1-17, which possesses the most typical characteristics of PDLSCs. Confocal co-localization experiments revealed the internalization of recombinant amelogenin (rM180) via binding to cell surface Grp78, and the endocytosis was inhibited by the silencing of Grp78 in 1-17 cells. Microarray analysis indicated that rM180 and Grp78 regulate the expression profiles of cell migration-associated genes in 1-17 cells. Moreover, Grp78 overexpression enhanced rM180-induced cell migration and adhesion without affecting cell proliferation, while silencing of Grp78 diminished these activities. Finally, binding of rM180 to Grp78 promoted the formation of lamellipodia, and the simultaneous activation of Rac1 was also demonstrated by NSC23766, a widely accepted Rac1 inhibitor. These results suggest that Grp78 is essential for enhancing amelogenin-induced migration in 1-17 cells. The biological interaction of amelogenin with Grp78 offers significant therapeutic potential for understanding the biological components and specific functions involved in the signal transduction of amelogenin-induced periodontal tissue regeneration.

Mutation of Spry2 induces proliferation and differentiation of osteoblasts but inhibits proliferation of gingival epithelial cells.

Sprouty was identified as an inhibitor of the fibroblast growth factor (FGF) receptor, and Sprouty2 (Spry2) functions as a negative regulator of receptor tyrosine kinase signaling. In this study, we investigated how inhibition of Spry2 affects osteoblasts and gingival epithelial cells in periodontal tissue regeneration in vitro. Transduction of a dominant-negative mutant of Spry2 (Y55A-Spry2) enhanced basic fibroblast growth factor (bFGF)- and epidermal growth factor (EGF)-induced ERK activation in MC3T3-E1 osteoblastic cells. In contrast, it decreased their activation in GE1 cells. Consistent with these observations, Y55A-Spry2 increased osteoblast proliferation with bFGF and EGF stimulation, whereas the proliferation of Y55A-Spry2-introduced GE1 cells was decreased via the ubiquitination and degradation of EGF receptors (EGFRs). In addition, Y55A-Spry2 caused upregulation of Runx2 expression and downregulation of Twist, a negative regulator of Runx2, with treatment of bFGF and EGF, resulting in enhanced osteoblastogenesis accompanied by alkaline phosphatase activation and osteocalcin expression in MC3T3-E1 cells. These data suggest that suppression of Spry2 expression induces proliferation and differentiation of osteoblastic cells after the addition of a bFGF and EGF cocktail but inhibits proliferation in gingival epithelial cells. These in vitro experiments may provide a molecular basis for novel therapeutic approaches in periodontal tissue regeneration. Taken together, our study proposes that combined application of an inhibitor for tyrosine 55 of Spry2, bFGF, and EGF may effectively allow alveolar bone growth and block the ingrowth of gingival epithelial cells toward bony defects, biologically mimicking a barrier effect in guided tissue regeneration, with in vivo investigation in the future.

Identification of novel amelogenin-binding proteins by proteomics analysis.

Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology. It is used in periodontal surgery to regenerate cementum, periodontal ligament, and alveolar bone. However, the precise molecular mechanisms underlying periodontal regeneration are still unclear. In this study, we investigated the proteins bound to amelogenin, which are suggested to play a pivotal role in promoting periodontal tissue regeneration. To identify new molecules that interact with amelogenin and are involved in osteoblast activation, we employed coupling affinity chromatography with proteomic analysis in fractionated SaOS-2 osteoblastic cell lysate. In SaOS-2 cells, many of the amelogenin-interacting proteins in the cytoplasm were mainly cytoskeletal proteins and several chaperone molecules of heat shock protein 70 (HSP70) family. On the other hand, the proteomic profiles of amelogenin-interacting proteins in the membrane fraction of the cell extracts were quite different from those of the cytosolic-fraction. They were mainly endoplasmic reticulum (ER)-associated proteins, with lesser quantities of mitochondrial proteins and nucleoprotein. Among the identified amelogenin-interacting proteins, we validated the biological interaction of amelogenin with glucose-regulated protein 78 (Grp78/Bip), which was identified in both cytosolic and membrane-enriched fractions. Confocal co-localization experiment strongly suggested that Grp78/Bip could be an amelogenin receptor candidate. Further biological evaluations were examined by Grp78/Bip knockdown analysis with and without amelogenin. Within the limits of the present study, the interaction of amelogenin with Grp78/Bip contributed to cell proliferation, rather than correlate with the osteogenic differentiation in SaOS-2 cells. Although the biological significance of other interactions are not yet explored, these findings suggest that the differential effects of amelogenin-derived osteoblast activation could be of potential clinical significance for understanding the cellular and molecular bases of amelogenin-induced periodontal tissue regeneration.

Zoledronic acid enhances lipopolysaccharide-stimulated proinflammatory reactions through controlled expression of SOCS1 in macrophages.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious side effect of nitrogen-containing bisphosphonate (NBP) use. Many studies have shown that BRONJ is limited to the jawbone and does not occur in the other bones. We hypothesized that BRONJ is related to local bacterial iections and involves the innate immune system. To examine the relationship between BRONJ and innate immunity, we examined the effects of NBPs on macrophages, one of the important cell types in innate immunity. The expression of toll-like receptor-4 (TLR4) in cells after pretreatment with zoledronic acid (ZOL) did not considerably differ from that in untreated control cells. However, cytokine levels and nitric oxide (NO) production increased after pretreatment with ZOL. Furthermore, ZOL induced NF-κB activation by enhancing IκB-α degradation. Lipopolysaccharide (LPS)-induced apoptosis also increased after pretreatment with ZOL. This effect was mediated by a reduction of suppressor of cytokine signaling-1 (SOCS1), which is a negative regulator of myeloid differentiation primary response gene 88 (MyD 88)-dependent signaling. These results suggest that ZOL induced excessive innate immune response and proinflammatory cytokine production and that these processes may be involved in the bone destruction observed in BRONJ.

Sprouty2 controls proliferation of palate mesenchymal cells via fibroblast growth factor signaling.

Cleft palate is one of the most common craniofacial deformities. The fibroblast growth factor (FGF) plays a central role in reciprocal interactions between adjacent tissues during palatal development, and the FGF signaling pathway has been shown to be inhibited by members of the Sprouty protein family. In this study, we report the incidence of cleft palate, possibly caused by failure of palatal shelf elevation, in Sprouty2-deficient (KO) mice. Sprouty2-deficient palates fused completely in palatal organ culture. However, palate mesenchymal cell proliferation estimated by Ki-67 staining was increased in Sprouty2 KO mice compared with WT mice. Sprouty2-null palates expressed higher levels of FGF target genes, such as Msx1, Etv5, and Ptx1 than WT controls. Furthermore, proliferation and the extracellular signal-regulated kinase (Erk) activation in response to FGF was enhanced in palate mesenchymal cells transfected with Sprouty2 small interfering RNA. These results suggest that Sprouty2 regulates palate mesenchymal cell proliferation via FGF signaling and is involved in palatal shelf elevation.

The Sprouty-related protein, Spred-1, localizes in a lipid raft/caveola and inhibits ERK activation in collaboration with caveolin-1.

Caveolin-1 (Cav-1) has been suggested to function as a negative regulator of mitogen-stimulated proliferation and the Ras-p42/44 ERK (MAP kinase) pathway in a variety of cell types. However, the molecular basis of this suppression has not been clarified. Spred/Sprouty family proteins are also negative regulators of the ERK pathway by interacting with Raf-1. The Spred/Sprouty family proteins contain a cysteine-rich (CR) domain at the C-terminus, which is thought to be palmitoylated like Cav-1 and necessary for membrane anchoring. In this study, we demonstrated that Spred-1 localized in cholesterol-rich membrane raft/caveola fractions and interacted with Cav-1. To clarify the biological effect of Cav-1/Spred-1 interaction, we used hematopoietic cells that lacked expression of caveolins but expressed Spred-1. Forced expression of Cav-1 suppressed SCF- and IL-3-induced proliferation and ERK activation. Furthermore, forced expression of exogenous Spred-1 in Cav-1-expressing cells further suppressed proliferation and ERK activation. These data suggest that Spred-1 inhibits ERK activation in collaboration with Cav-1.

Loss of mammalian Sprouty2 leads to enteric neuronal hyperplasia and esophageal achalasia.

We report here that loss of the Sprouty2 gene (also known as Spry2) in mice resulted in enteric nerve hyperplasia, which led to esophageal achalasia and intestinal pseudo-obstruction. Glial cell line-derived neurotrophic factor (GDNF) induced hyperactivation of ERK and Akt in enteric nerve cells. Anti-GDNF antibody administration corrected nerve hyperplasia in Sprouty2-deficient mice. We show Sprouty2 to be a negative regulator of GDNF for the neonatal development or survival of enteric nerve cells.

Spred-1 negatively regulates interleukin-3-mediated ERK/mitogen-activated protein (MAP) kinase activation in hematopoietic cells.

Sprouty/Spred family proteins have been identified as negative regulators of growth factor-induced ERK/mitogen-activated protein (MAP) kinase activation. However, it has not been clarified whether these proteins regulate cytokine-induced ERK activity. We found that Spred-1 is highly expressed in interleukin-3 (IL-3)-dependent hematopoietic cell lines and bone marrow-derived mast cells. To investigate the roles of Spred-1 in hematopoiesis, we expressed wild-type Spred-1 and a dominant negative form of Spred-1, DeltaC-Spred, in IL-3- and stem cell factor (SCF)-dependent cell lines as well as hematopoietic progenitor cells from mouse bone marrow by retrovirus gene transfer. In IL-3-dependent Ba/F3 cells expressing c-kit, forced expression of Spred-1 resulted in a reduced proliferation rate and ERK activation in response to not only SCF but also IL-3. In contrast, DeltaC-Spred augmented IL-3-induced cell proliferation and ERK activation. Wild-type Spred-1 inhibited colony formation of bone marrow cells in the presence of cytokines, whereas DeltaC-Spred-1 expression enhanced colony formation. Augmentation of ERK activation and proliferation in response to IL-3 was also observed in Spred-1-deficient bone marrow-derived mast cells. These data suggest that Spred-1 negatively regulates hematopoiesis by suppressing not only SCF-induced but also IL-3-induced ERK activation.

Model analysis of difference between EGF pathway and FGF pathway.

The difference in time course of Ras and mitogen activated protein kinase (MAPK) cascade by different growth factors is considered to be the cause of different cellular responses. We have developed the computer simulation of Ras-MAPK signal transduction pathway containing newly identified negative feedback system, Sprouty, and adaptor molecules. Unexpectedly, negative feedback system did not profoundly affect time course of MAPK activation. We propose the key role of fibroblast growth factor receptor substrate 2 (FRS2) in NGF/FGF pathway for sustained MAPK activation. More Grb2-SOS complexes were recruited to the plasma membrane by binding to membrane-bound FRS2 in FGF pathway than in EGF pathway and caused sustained activation of ERK. The EGF pathway with high concentration of EGF receptor also induced sustained MAPK activation, which is consistent with the results in the PC12 cell overexpressing the EGF receptors. The simulated time courses of FRS2 knock-out cells were consistent with those of the reported experimental results.