Expression Of Spred-1 Research Articles

Astragaloside IV promotes exosome secretion of endothelial progenitor cells to regulate PIK3R2/SPRED1 signaling and inhibit pyroptosis of diabetic endothelial cells

Background aimsTreating chronic non-healing diabetic wounds and achieving complete skin regeneration has always been a critical clinical challenge. MethodsIn order to address this issue, researchers conducted a study aiming to investigate the role of miR-126-3p in regulating the downstream gene PIK3R2 and promoting diabetic wound repair in endothelial progenitor cell (EPC)–derived extracellular vesicles. The study involved culturing EPCs with astragaloside IV, transfecting them with miR-126-3p inhibitor or mock plasmid, interfering with high glucose-induced damage in human umbilical vein endothelial cells (HUVECs) and treating diabetic skin wounds in rats. ResultsThe healing of rat skin wounds was observed through histological staining. The results revealed that treatment with miR-126-3p–overexpressing EPC-derived extracellular vesicles accelerated the healing of rat skin wounds and resulted in better tissue repair with slower scar formation. In addition, the transfer of EPC-derived extracellular vesicles with high expression of miR-126-3p to high glucose-damaged HUVECs increased their proliferation and invasion, reduced necrotic and apoptotic cell numbers and improved tube formation. In this process, the expression of angiogenic factors vascular endothelial growth factor (VEGF)A, VEGFB, VEGFC, basic fibroblast growth factor and Ang-1 significantly increased, whereas the expression of caspase-1, NRLP3, interleukin-1β, inteleukin-18, PIK3R2 and SPRED1 was suppressed. Furthermore, miR-126-3p was able to target and inhibit the expression of the PIK3R2 gene, thereby restoring the proliferation and migration ability of high glucose-damaged HUVEC. ConclusionsIn summary, these research findings demonstrate the important role of miR-126-3p in regulating downstream genes and promoting diabetic wound repair, providing a new approach for treating chronic non-healing diabetic wounds.

Transplantation of endothelial progenitor cells overexpressing mir-126-3p improves vascular repair in a diabetic rat model.

Transplantation of endothelial progenitor cells overexpressing mir-126-3p improves vascular repair in a diabetic rat model.

SPOCK2 and SPRED1 function downstream of EZH2 to impede the malignant progression of lung adenocarcinoma in vitro and in vivo

Enhancer of zeste homolog 2 (EZH2) is an important epigenetic regulator, and is associated with the malignant progression of lung cancer. However, the mechanisms of EZH2 on lung adenocarcinoma (LUAD) remain unclear. The relationship between EZH2 and SPOCK2 or SPRED1 was confirmed by dual-luciferase reporter assay. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were analyzed to examine the expression of SPOCK2 and SPRED1 and their prognostic values of LUAD. The effects of SPOCK2 and SPRED1 on the biological characters of LUAD cells were identified on functional assays in vitro and in vivo. Our results showed that EZH2 suppressed the expression and transcriptional activity of SPOCK2 and SPRED1, and these effects were reversed by the EZH2 inhibitor, Tazemetostat. SPOCK2 and SPRED1 were expressed at low levels in LUAD patients, and a high expression level of SPOCK2 or SPRED1 predicted better survival. Moreover, overexpression of SPOCK2 or SPRED1 could inhibit tumoral proliferation, migration ratio, and invasion activity in vitro as well as retard tumor growth in vivo. However, EZH2 elevation could rescue these impacts and accelerate LUAD progression. Our findings reveal that SPOCK2 and SPRED1 are epigenetically suppressed by EZH2 and may act as novel regulators to inhibit the proliferation, migration, and invasion of LUAD cells.

MiR-126-3p-loaded small extracellular vesicles secreted by urine-derived stem cells released from a phototriggered imine crosslink hydrogel could enhance vaginal epithelization after vaginoplasty

BackgroundDue to the large area and deep width of the artificial neovagina after vaginoplasty, it takes a considerable amount of time to achieve complete epithelization of the neovagina. Currently, the clinical therapies for vaginal epithelization after vaginoplasty are still dissatisfactory. Recent studies showed that small extracellular vesicles (sEVs) derived from stem cells could accelerate wound epithelization. The sustained release of sEVs from optimized hydrogels may be a promising strategy to accelerate vaginal epithelization after vaginoplasty.MethodsThe efficacy of phototriggered imine crosslink hydrogels (piGEL) containing sEVs derived from human urine-derived stem cells (hUSC-sEVs, piGEL-sEVs) on vaginal mucosa defects in rabbits was assessed by wound closure rates, histological analysis and immunofluorescence staining analysis. Cell counting kit-8, 5-ethynyl-2′-deoxyuridine and scratch wound assays were performed to assess the effects of hUSC-sEVs on the proliferation and migration ability of vaginal epithelial cells (VK2/E6E7). Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to test the expression of epithelial differentiation markers in VK2 cells. Moreover, a microRNA (miRNA) microarray was used to find hUSC-sEVs-specific miRNAs that potentially affected the proliferation, migration and differentiation ability of VK2 cells.ResultsThe in vitro release profile revealed that the piGEL could ensure sustained release of hUSC-sEVs. The in vivo results showed that piGEL-sEVs effectively promoted epithelization and angiogenesis of vaginal mucosa defects in rabbits. According to miRNA microarray and qRT-PCR results, miR-126-3p might be the crucial molecule among the various miRNAs contained in hUSC-sEVs. The data showed that hUSC-sEVs promoted the migration and differentiation of VK2 cells by delivering miR-126-3p to suppress the expression of Spred1 and PIK3R2, thereby activating the ERK1/2 and ATK signaling pathways.ConclusionThe results indicated that piGEL-sEVs could be a novel promising approach for enhancing the epithelization of the neovagina after vaginoplasty and provided useful data for understanding the underlying mechanism of the effect of hUSC-sEVs on epithelization.

Angiogenesis in diabetic mouse model with critical limb ischemia; cell and gene therapy.

Critical limb ischemia (CLI) is the most severe manifestation of peripheral artery disease that diabetes mellitus is one of its major risk factors. MiR-126 as an endothelial cells specific miRNA plays a main role in angiogenesis. The objective of this study was to find a promising treatment by increasing therapeutic potential of adipose tissue mesenchymal stem cells (AT-MSCs) with microRNA-126 in diabetic mouse model with critical limb ischemia. AT-MSCs were isolated from male C57BL/6 mouse and characterized. The cells were infected with miR-126 recombinant lentiviral vectors. Diabetes mellitus type 1 was induced and CLI was created in the animals. Animals were divided in different groups to receive PBS, MSCs, miR-126, and MSCmiR-126 and after the experiment, behavioural tests, cell survival, real-time PCR, and histopathological analysis were assessed. The results of function scores, VEGF-A level, and histopathology data demonstrated that the miR-126 treated group was better than PBS and MSCs groups. The expression of PIK3R2 and SPRED1 were decreased in miR-126 group compared to the control group. Our results showed that MSCsmiR-126 can live longer than MSCs in the gastrocnemius muscle. We conclude that mice treated with MSCsmiR-126 in functional tests showed better results and also the expression of VEGF-A and Microvessel density in them were higher than other groups. This study suggested that AT-MSCs overexpressing miR-126 could be an efficient therapeutic approach for angiogenesis in CLI with diabetes by downregulating SPRED1 and PIK3R2 and increasing secretion of angiogenic cytokines which can prolong the MSC survival.

HIF-1α-induced up-regulation of microRNA-126 contributes to the effectiveness of exercise training on myocardial angiogenesis in myocardial infarction rats.

Exercise training (ET) is a non‐drug natural rehabilitation approach for myocardial infarction (MI). Among the numerous beneficial effects of ET, myocardial angiogenesis is indispensable. In the present study, we investigated the role and mechanism of HIF‐1α and miR‐126 in ET‐induced MI myocardial angiogenesis which may provide new insights for MI treatment. Rat model of post‐MI and human umbilical vein endothelial cells (HUVECs) were employed for our research. Histomorphology, immunohistochemistry, quantitative real‐time PCR, Western blotting and small‐interfering RNA (siRNA) transfection were applied to evaluate the morphological, functional and molecular mechanisms. In vivo results showed that 4‐week ET could significantly increase the expression of HIF‐1α and miR‐126 and reduce the expression of PIK3R2 and SPRED1, while 2ME2 (HIF‐1α inhibitor) partially attenuated the effect of ET treatment. In vitro results showed that HIF‐1α could trigger expression of miR‐126 in HUVECs in both normoxia and hypoxia, and miR‐126 may be involved in the tube formation of HUVECs under hypoxia through the PI3K/AKT/eNOS and MAPK signalling pathway. In conclusion, we revealed that HIF‐1α, whose expression experiences up‐regulation during ET, could function as an upstream regulator to miR‐126, resulting in angiogenesis promotion through the PI3K/AKT/eNOS and MAPK signalling pathway and subsequent improvement of the MI heart function.

A Study on the Expression of SPRED1 and PBRM1 (Baf180) and their Clinical Significances in Patients with Gastric Cancer.

To evaluate SPRED1 and PBRMl expression in patients with gastric cancer and determine the biological relationships of SPRED1 and PBRM1 with the occurrence and development of gastric cancer. Tissue specimens of patients with gastric cancer at Jingzhou First People's Hospital were gathered from April 2016 to August 2018. SPRED1 and PBRMl (Baf180) protein expression levels were detected in the excised cancerous tissues and normal tissues (control group) of 80 patients with gastric cancer by immunohistochemical methods. The positive rates of SPRED1 and PBRMl protein expression in gastric cancer tissues were 55% and 75%, respectively. The positive rates of SPRED1 and PBRMl protein expression in the normal tissues without cancer were 84.6% and 92.3%, respectively. The expression in gastric cancer tissues was significantly lower than that of the control group (p < 0.05). Positive SPRED1 and PBRMl protein expression was related to histological type, depth of infiltration, presence of lymphatic metastasis, pathological grade, and clinical TNM phase (p < 0.05). SPRED1 expression and PBRMl expression were positively correlated. The expression of SPRED1 and PBRMl in gastric cancer tissues is low, unrelated to age and declines with increasing pathological grade and clinical phase of the gastric cancer tissues. SPRED1 and PBRMl expression may be related to the biological behavior of tumors, and the two may have a synergistic effect.

Danhong injection enhances angiogenesis after myocardial infarction by activating MiR-126/ERK/VEGF pathway

Background/aimDanhong injection (DHI) is a Chinese drug used for relieving cardiovascular diseases. This study aimed to identify the effect and mechanism of action of DHI on post-infarct angiogenesis, especially the epigenetic regulation of angiogenesis. MethodsA myocardial infarction (MI) mouse model was induced by ligating the left anterior descending coronary artery. A 4-week daily treatment with or without DHI via intraperitoneal injection was started immediately following MI. The changes in cardiac function, pathology, and angiogenesis following MI were measured by echocardiography and immunostaining. Matrigel tube formation and scratch wound assays were used to evaluate the effect of DHI on the proliferation and migration of hypoxic human umbilical vein endothelial cells (HUVECs). The expression of miR-126, Spred-1, and angiogenesis-related mRNAs was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of related proteins and the phosphorylated levels of extracellular signal–regulated kinase (ERK) and protein kinase B were detected by Western blot analysis. The loss-of-function study was performed using antagomir-126. ResultsThe DHI-treated mice had significantly reduced infarct area, improved ejection fraction, and increased capillary density 4 weeks after MI. Also, DHI promoted the proliferation and migration of hypoxic HUVECs. The qRT-PCR and Western blot analysis revealed that DHI intervention upregulated miR-126, suppressed Spred-1 expression, and activated the ERK pathway, but not the Akt pathway. The loss-of-function study showed the blockade of the pro-angiogenic effect of DHI by antagomir-126 involving the ERK/vascular endothelial growth factor (VEGF) pathway. ConclusionDHI enhanced post-infarct angiogenesis after MI by activating the miR-126/ERK/VEGF pathway.

Lipoxin A4 stimulates endothelial miR-126-5p expression and its transfer via microvesicles.

The proresolution lipid mediator lipoxin (LX)A4 bestows protective bioactions on endothelial cells. We examined the impact of LXA4 on transcellular endothelial signaling via microRNA (miR)-containing microvesicles. We report LXA4 inhibition of MV release by TNF-α-treated HUVECs, associated with the down-regulation of 18 miR in endothelial microvesicles (EMVs) and the up-regulation of miR-126-5p, both in HUVECs and in EMVs. LXA4 up-regulated miR-126-5p by ∼5-fold in HUVECs and promoted a release of microvesicles (LXA4-EMVs) that enhanced miR-126-5p by ∼7-fold in recipient HUVECs. In these cells, LXA4-EMVs abrogated the up-regulation of VCAM-1, induced in recipient HUVECs by EMVs released by untreated or TNF-α-treated HUVECs. LXA4-EMVs also reduced by ∼40% the expression of SPRED1, which we validated as an miR-126-5p target, whereas they stimulated monolayer repair in an in vitro wound assay. This effect was lost when the EMVs were depleted of miR-126-5p. These results provide evidence that changes in miR expression and microvesicle packaging and transfer represent a mechanism of action of LXA4, which may be relevant in vascular biology and inflammation.-Codagnone, M., Recchiuti, A., Lanuti, P., Pierdomenico, A. M., Cianci, E., Patruno, S., Mari, V. C., Simiele, F., Di Tomo, P., Pandolfi, A., Romano, M. Lipoxin A4 stimulates endothelial miR-126-5p expression and its transfer via microvesicles.

GW27-e0405 Study on the compatibility of Blood Activation and Qi Supplement prescription(BAQS) on angiogenesis regulated by the expression of miR-126 and Spred1 in rats after AMI

GW27-e0405 Study on the compatibility of Blood Activation and Qi Supplement prescription(BAQS) on angiogenesis regulated by the expression of miR-126 and Spred1 in rats after AMI

SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling.

The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is tightly controlled by negative feedback regulators, such as the tumor suppressor SPRED1. The SPRED1 gene also carries loss-of-function mutations in the RASopathy Legius syndrome. Growth factor stimulation translocates SPRED1 to the plasma membrane, triggering its inhibitory activity. However, it remains unclear whether SPRED1 there acts at the level of Ras or Raf. We show that pharmacological or galectin-1 (Gal-1)-mediated induction of B- and C-Raf-containing dimers translocates SPRED1 to the plasma membrane. This is facilitated in particular by SPRED1 interaction with B-Raf and, via its N terminus, with Gal-1. The physiological significance of these novel interactions is supported by two Legius syndrome-associated mutations that show diminished binding to both Gal-1 and B-Raf. On the plasma membrane, SPRED1 becomes enriched in acidic membrane domains to specifically perturb membrane organization and extracellular signal-regulated kinase (ERK) signaling of active K-ras4B (here, K-ras) but not H-ras. However, SPRED1 also blocks on the nanoscale the positive effects of Gal-1 on H-ras. Therefore, a combinatorial expression of SPRED1 and Gal-1 potentially regulates specific patterns of K-ras- and H-ras-dependent signaling output. More broadly, our results open up the possibility that related SPRED and Sprouty proteins act in a similar Ras and Raf isoform-specific manner.

Inhibition of microRNA-126 promotes the expression of Spred1 to inhibit angiogenesis in hepatocellular carcinoma after transcatheter arterial chemoembolization: in vivo study.

MicroRNA-126 (miR-126) has been found to promote angiogenesis, but the underlying mechanisms are still unclear. So, we conducted this study to explore the effect of miR-126 expression on angiogenesis in hepatocellular carcinoma (HCC) after transcatheter arterial chemoembolization (TACE). The expression levels of miR-126 and sprouty-related, EVH1 domain containing protein (Spred)1 in surgically resected HCC tissue, HCC tissue with TACE + operation, and tumor-adjacent tissues were determined by quantitative real-time polymerase chain reaction. The expression levels of miR-126, Spred1, and vascular endothelial growth factor were found by quantitative real-time polymerase chain reaction and Western blot. The microvessel density (MVD) of tumor tissues was determined by immunohistochemical staining. The miR-126 and Spred1 expressions in HCC tissue with TACE + operation were elevated and decreased, respectively, as compared to those in surgically resected HCC tissues and tumor-adjacent tissues (all P<0.001), which indicated that the expression of Spred1 was negatively correlated with miR-126 (P<0.001, r=−0.6224). Based on the bioinformatics analysis and luciferase reporter gene activity detection, Spred1 was found to target miR-126 (P<0.001). Inhibition of miR-126 expression reduces the degree of weight loss and tumor size in TACE model rats. The MVD in TACE + operation group was increased compared to that in the control group; inhibition of miR-126 expression had a reversal effect, to a certain extent, on MVD increase after TACE (all P<0.05). Inhibition of miR-126 expression increased Spred1 expression and decreased vascular endothelial growth factor expression (P<0.01). In summary, this study unveiled the potential mechanism by which miR-126 regulates angiogenesis in HCC tissues through embolization treatment by targeting Spred1, and also showed that the feasibility of TACE with the miR-126 inhibitor has a certain value in the medical treatment of HCC.

Expression of Sproutys and SPREDs is decreased during lung branching morphogenesis in nitrofen-induced pulmonary hypoplasia

Pulmonary hypoplasia (PH) is a life-threatening condition associated with congenital diaphragmatic hernia (CDH), characterized by defective lung development. Sproutys and Sprouty-related proteins (SPREDs) play a key role in lung branching morphogenesis through modification of epithelial-mesenchymal interactions. During the pseudoglandular stage, Sproutys are highly expressed in distal airway epithelium, while SPREDs within the surrounding mesenchyme. Sprouty2/4 knockouts show severe defects in branching morphogenesis with reduced number of distal airways. SPRED-1 and SPRED-2 are strongly expressed in regions of new airway formation, highlighting their important function in branching pattern. We hypothesized that expression of Sprouty2, Sprouty4, SPRED-1 and SPRED-2 is decreased during lung branching morphogenesis in nitrofen-induced PH. Timed-pregnant rats received either nitrofen or vehicle on E9.5. On E15.5 (n = 16), fetal lungs were micro-dissected and divided into controls and PH, while on E18.5 (n = 24) groups were: control, PH without CDH [CDH(-)], and PH with CDH [CDH(+)]. Pulmonary gene expression levels of Sprouty2, Sprouty4, SPRED-1 and SPRED-2 were analyzed by qRT-PCR. Immunohistochemistry was performed to evaluate protein expression/distribution. On E18.5, relative mRNA expression levels of Sprouty2, Sprouty4, SPRED-1 and SPRED-2 were significantly decreased in CDH(-) and CDH(+) groups compared to controls (P < 0.05). Immunoreactivity of Sprouty2, Sprouty4, SPRED-1 and SPRED-2 was markedly diminished on E18.5 in nitrofen-induced PH. Decreased expression of Sproutys and SPREDs during the terminal pseudoglandular stage may disrupt lung branching morphogenesis by interfering with epithelial-mesenchymal interactions contributing to PH.

MicroRNA-126 expression is decreased in cultured primary chicken hepatocytes and targets the sprouty-related EVH1 domain containing 1 mRNA

The microRNA-126 (miR-126) is a miRNA expressed in highly vascularized tissues, and it is believed to play a role in angiogenesis by repressing sprouty-related EVH1 domain containing 1 (Spred1). In the current study, we determined the expression pattern of chicken miR-126 (gga-miR-126) and predicted and validated its target genes. The quantitative reverse-transcription (qRT) PCR analysis showed that miR-126 was expressed in various chicken tissues with the highest level in lung. In liver, the expression level of miR-126 increased from 0 to 7 wk of age. The expression of miR-126 in primary chicken hepatocytes decreased with culturing. A miR-126 binding site was predicted in the 3' UTR (untranslated region) of chicken Spred1. Dual-luciferase reporter assays indicated that miR-126 could bind to the predicted site to repress the expression of Spred1. These data validate Spred1 as a target gene of chicken miR-126. These results will help further understand the function and regulation of miR-126 and Spred1 in chickens.

TGF-β modulates the functionality of tumor-infiltrating CD8+ T cells through effects on TCR signaling and Spred1 expression

This study demonstrates that CD8+ T cells in the tumor microenvironment display reduced functionality and hyporesponsiveness. TGF-beta contributed markedly to the tumor-infiltrating CD8+ T cells' (TILs) reduced functionality, which could be reversed using a small molecule TGF-beta inhibitor. Upon T-cell receptor (TCR) activation, the activation of ITK and ERK kinases were reduced in CD8+ TILs, as compared to splenic CD8+ T cells: TGF-beta inhibitor could reverse this phenomenon. This study demonstrates for the first time the association of the Spred-1 gene, an inhibitor of the Ras/MAPK pathway, with CD8+ TILs and TGF-beta activity. Spred-1 was upregulated in CD8+ TILs and TGF-beta enhanced the expression of Spred-1 in effector/memory CD8+ T cells and not in rested/memory CD8+ T cells. Based on these findings, this study supports the hypothesis that TGF-beta mediates an inhibitory mechanism on CD8+ TILs involving TCR-signaling blockade and the upregulation of Spred-1, thus implicating Spred-1 as a potential new target for future anti-tumor immune studies.

MiR-126 Regulates Angiogenic Signaling and Vascular Integrity

Precise regulation of the formation, maintenance, and remodeling of the vasculature is required for normal development, tissue response to injury, and tumor progression. How specific microRNAs intersect with and modulate angiogenic signaling cascades is unknown. Here, we identified microRNAs that were enriched in endothelial cells derived from mouse embryonic stem (ES) cells and in developing mouse embryos. We found that miR-126 regulated the response of endothelial cells to VEGF. Additionally, knockdown of miR-126 in zebrafish resulted in loss of vascular integrity and hemorrhage during embryonic development. miR-126 functioned in part by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein SPRED1 and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2/p85-beta). Increased expression of Spred1 or inhibition of VEGF signaling in zebrafish resulted in defects similar to miR-126 knockdown. These findings illustrate that a single miRNA can regulate vascular integrity and angiogenesis, providing a new target for modulating vascular formation and function.

The Endothelial-Specific MicroRNA miR-126 Governs Vascular Integrity and Angiogenesis

Endothelial cells play essential roles in maintenance of vascular integrity, angiogenesis, and wound repair. We show that an endothelial cell-restricted microRNA (miR-126) mediates developmental angiogenesis in vivo. Targeted deletion of miR-126 in mice causes leaky vessels, hemorrhaging, and partial embryonic lethality, due to a loss of vascular integrity and defects in endothelial cell proliferation, migration, and angiogenesis. The subset of mutant animals that survives displays defective cardiac neovascularization following myocardial infarction. The vascular abnormalities of miR-126 mutant mice resemble the consequences of diminished signaling by angiogenic growth factors, such as VEGF and FGF. Accordingly, miR-126 enhances the proangiogenic actions of VEGF and FGF and promotes blood vessel formation by repressing the expression of Spred-1, an intracellular inhibitor of angiogenic signaling. These findings have important therapeutic implications for a variety of disorders involving abnormal angiogenesis and vascular leakage.

Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors

Aberrant activation of the Ras/Raf-1/extracellular-regulated kinase (ERK) pathway has been shown to be involved in the progression of human hepatocellular carcinoma (HCC). However, the mechanism of dysregulation of ERK activation is poorly understood. Recently, we identified Sprouty-related protein with Ena/vasodilator-stimulated phosphoprotein homology-1 domain (Spred) as a physiological inhibitor of the Ras/Raf-1/ERK pathway. In this study, we found that the expression levels of Spred-1 and -2 in human HCC tissue were frequently decreased, comparing with those in adjacent non-tumorous tissue. Moreover, Spred expression levels in HCC tissue were inversely correlated with the incidence of tumor invasion and metastasis. Forced expression of Spred-1 inhibited HCC cell proliferation in vitro and in vivo, which was associated with reduced ERK activation. Spred-1 overexpression also reduced the secretion of matrix metalloproteinase-9 (MMP-9) and MMP-2, which play important roles in tumor invasion and metastasis. In addition, Spred-1 inhibited growth factor-mediated HCC cell motility. These data indicate that the reduction of Spred expression in HCC is one of the causes of the acquisition of malignant features. Thus, Spred could be not only a novel prognostic factor but also a new therapeutic target for human HCC.

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.

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.