Activated Rap1A Induces Osteoblastic Differentiation and Cell Adhesion

김혜선,전택중 조선대학교 기초과학연구원 2016 조선자연과학논문집 Vol.9 No.3

Rap1 is a key regulator of cell adhesion and migration. Although increasing evidence indicates that the Rap1 signaling pathway is involved in the process of bone remodeling, the mechanism by which Rap1 regulates osteoblastic differentiation and cell adhesion remains unknown. Here, we investigated the morphological characteristics and osteoblastic differentiation of cells expressing constitutively activated form of Rap1A (Rap1ACA) or Rap1 GTPase activating protein Rap1GAP and found that activated Rap1 induces osteoblastic differentiation and cell adhesion as well as cell spreading. When osteoblastic differentiation was induced, Rap1ACA cells showed considerably higher levels of calcium deposits than the wild-type and Rap1GAP-overexpressing cells did. Rap1ACA cells showed increased spreading and size, as well as strong cell adhesion and significantly decreased growth rates. F-actin staining using phalloidin revealed several thin thread-like filopodia around the protrusions in Rap1ACA cells, which possibly contribute to the increased cell adhesion.

Activated Rap1A Induces Osteoblastic Differentiation and Cell Adhesion

Kim, Hyeseon,Jeon, Taeck J. The Basic Science Institute Chosun University 2016 조선자연과학논문집 Vol.9 No.3

Rap1 is a key regulator of cell adhesion and migration. Although increasing evidence indicates that the Rap1 signaling pathway is involved in the process of bone remodeling, the mechanism by which Rap1 regulates osteoblastic differentiation and cell adhesion remains unknown. Here, we investigated the morphological characteristics and osteoblastic differentiation of cells expressing constitutively activated form of Rap1A (Rap1ACA) or Rap1 GTPase activating protein Rap1GAP and found that activated Rap1 induces osteoblastic differentiation and cell adhesion as well as cell spreading. When osteoblastic differentiation was induced, Rap1ACA cells showed considerably higher levels of calcium deposits than the wild-type and Rap1GAP-overexpressing cells did. Rap1ACA cells showed increased spreading and size, as well as strong cell adhesion and significantly decreased growth rates. F-actin staining using phalloidin revealed several thin thread-like filopodia around the protrusions in Rap1ACA cells, which possibly contribute to the increased cell adhesion.

Regulation of Actin Cytoskeleton by Rap1 Binding to RacGEF1

Hyemin Mun,전택중 한국분자세포생물학회 2012 Molecules and cells Vol.34 No.1

Rap1 is rapidly and transiently activated in response to chemoattractant stimulation and helps establish cell polarity by locally modulating cytoskeletons. Here, we investigated the mechanisms by which Rap1 controls actin cytoskeletal reorganization in Dictyostelium and found that Rap1 interacts with RacGEF1 in vitro and stimulates F-actin polymerization at the sites where Rap1 is activated upon chemoattractant stimulation. Live cell imaging using GFP-coronin, a reporter for F-actin, demonstrates that cells expressing constitutively active Rap1 (Rap1CA) exhibit a high level of F-actin uniformly distributed at the cortex including the posterior and lateral sides of the chemotaxing cell. Examination of the localization of a PH-domain containing PIP3 reporter, PhdA-GFP, and the activation of Akt/Pkb and other Ras proteins in Rap1CA cells reveals that activated Rap1 has no effect on the production of PIP3 or the activation of Akt/Pkb and Ras proteins in response to chemoattractant stimulation. Rac family proteins are crucial regulators in actin cytoskeletal reorganization. In vitro binding assay using truncated RacGEF1 proteins shows that Rap1 interacts with the DH domain of RacGEF1. Taken together, these results suggest that Rap1-mediated F-actin polymerization probably occurs through the Rac signaling pathway by directly binding to RacGEF1.

Fission Yeast Rap1 Homolog Is a Telomere-specific silencing Factor and Interracts woth Taz1p

Park, Min Jung,Jang, Yeun Kyu,Choi, Eun Shik,Kim, Hyun Soo,Park, Sang Dai Korean Society of Molecular Biology 2002 Molecules and cells Vol.13 No.2

miR-101-3p/Rap1b signal pathway plays a key role in osteoclast differentiation after treatment with bisphosphonates

( Jie Li ),( You Li ),( Shengjie Wang ),( Hui Che ),( Jun Wu ),( Yongxin Ren ) 생화학분자생물학회 2019 BMB Reports Vol.52 No.9

Bisphosphonates are the mainstay of therapy worldwide for osteoporosis. However, bisphosphonates also have limitations. The objective of this study was to determine the role of miR-101-3p/Rap1b signal pathway in osteoclast differentiation after treatment with bisphosphonates. Our results revealed that miR-101-3p was an important regulator in bisphosphonates treated-osteoclasts. When miR-101-3p was down-regulated in bone marrow-derived macrophage-like cells (BMMs), the development of mature osteoclasts was promoted, and vice versa. However, alendronate decreased multinucleated cell number regardless of whether miR-101-3p was knocked down or over-expressed. TRAP activity assay confirmed the above results. Luciferase assay indicated that miR-101-3p was a negative regulator of Rap1b. Western blot analysis revealed that protein expression level of Rap1b in BMMs transfected with OV-miR-101-3p was lower than that in BMMs transfected with an empty vector. Rap1b overexpression increased TRAP-positive multinucleated cells, while Rap1b inhibition decreased the cell numbers. In vivo data showed that miR-101-3p inhibited osteoclast differentiation in ovariectomized mice while overexpressed of Rap1b blocked the differentiation. Taken together, our data demonstrate that miR-101-3p/Rap1b signal pathway plays a key role in osteoclast differentiation after treatment with bisphosphonates. [BMB Reports 2019; 52(9): 572-576]

NDR1-Dependent Regulation of Kindlin-3 Controls High-Affinity LFA-1 Binding and Immune Synapse Organization

Kondo, Naoyuki,Ueda, Yoshihiro,Kita, Toshiyuki,Ozawa, Madoka,Tomiyama, Takashi,Yasuda, Kaneki,Lim, Dae-Sik,Kinashi, Tatsuo American Society for Microbiology 2017 Molecular and cellular biology Vol.37 No.8

<P><B>ABSTRACT</B></P><P>Antigen-specific adhesion between T cells and antigen-presenting cells (APC) during the formation of the immunological synapse (IS) is mediated by LFA-1 and ICAM-1. Here, LFA-1–ICAM-1 interactions were measured at the single-molecule level on supported lipid bilayers. High-affinity binding was detected at low frequencies in the inner peripheral supramolecular activation cluster (SMAC) zone that contained high levels of activated Rap1 and kindlin-3. Rap1 was essential for T cell attachment, whereas deficiencies of ste20-like kinases, Mst1/Mst2, diminished high-affinity binding and abrogated central SMAC (cSMAC) formation with mislocalized kindlin-3 and vesicle transport regulators involved in T cell receptor recycling/releasing machineries, resulting in impaired T cell-APC interactions. We found that NDR1 kinase, activated by the Rap1 signaling cascade through RAPL and Mst1/Mst2, associated with and recruited kindlin-3 to the IS, which was required for high-affinity LFA-1/ICAM-1 binding and cSMAC formation. Our findings reveal crucial roles for Rap1 signaling via NDR1 for recruitment of kindlin-3 and IS organization.</P>

Chemoattractant-Mediated Rap1 Activation Requires GPCR/G Proteins

Cha, In-Jun,Lee, Sung-H.,Jeon, Taeck-J Korean Society for Molecular and Cellular Biology 2010 Molecules and cells Vol.30 No.6

Rap1 is rapidly activated upon chemoattractant stimulation and plays an important role in cell adhesion and cytoskeletal reorganization during chemotaxis. Here, we demonstrate that G-protein coupled receptors and G-proteins are essential for chemoattractant-mediated Rap1 activation in Dictyostelium. The rapid Rap1 activation upon cAMP chemoattractant stimulation was absent in cells lacking chemoattractant cAMP receptors cAR1/cAR3 or a subunit of the heterotrimeric G-protein complex G${\alpha}$2. Loss of guanylyl cyclases GCA/SGC or a cGMP-binding protein GbpC exhibited no effect on Rap1 activation kinetics. These results suggest that Rap1, a key regulator for the regulation of cytoskeletal reorganization during cell movement, is activated through the G-protein coupled receptors cAR1/cAR3 and G${\alpha}$2 proteins in a way independent of the cGMP signaling pathway.

MicroRNA-340-5p relieved chronic constriction injury-induced neuropathic pain by targeting Rap1A in rat model

Lu Gao,Xuehua Pu,Yujing Huang,Jing Huang 한국유전학회 2019 Genes & Genomics Vol.41 No.6

Objectives Neuropathic pain (NP) is one of the main challenges towards NP syndrome treatment. miR-340-5p exhibit different expression levels in NP models. Its effects on NP remained unclear. The objective of this study was to explore the potential regulation mechanisms of miR-340-5p in NP. Methods Rat model of chronic constriction injury (CCI) was established to induce NP in vivo. NP levels were assessed using mechanical withdrawal threshold (MWT). The inflammation response in CCI rats were determined by HE staining and ELISA assay. The target genes of miR-340-5p were verified by luciferase report assays. Results In CCI rats, level of miR-340-5p was down-regulated both in spinal cord tissues and isolated microglial cells. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were decreased in CCI rats, which were restored upon miR-340-5p overexpression. miR-340-5p overexpression also decreased inflammation as well as expression levels of COX- 2, IL-1β, TNF-α and IL-6 in CCI rats. Luciferase report assays revealed Rap1A was a target gene of miR-340-5p in the experimental model. Elevated miR-340-5p decreased Rap1A expression level in vitro and in vivo. Overexpression of Rap1A protein restored expression levels of COX-2, IL-1β, TNF-α and IL-6, reduced the PWT and PWL and increased inflammation response in CCI rats. Conclusion miR-340-5p alleviated CCI-induced NP by targeting Rap1A. miR-340-5p and Rap1A may be the potential treatment targets for NP therapeutics.

Factors forming the BRCA1-A complex orchestrate BRCA1 recruitment to the sites of DNA damage

Her, Joonyoung,Soo Lee, Nam,Kim, Yonghwan,Kim, Hongtae Oxford University Press 2016 Acta biochimica et biophysica Sinica Vol.48 No.7

<P>Sustaining genomic integrity is essential for preventing onset of cancers. Therefore, human cells evolve to have refined biological pathways to defend genetic materials from various genomic insults. DNA damage response and DNA repair pathways essential for genome maintenance are accomplished by cooperative executions of multiple factors including breast cancer type 1 susceptibility protein (BRCA1). BRCA1 is initially identified as an altered gene in the hereditary breast cancer patients. Since then, tremendous efforts to understand the functions of BRAC1 reveal that BRCA1 is found in distinct complexes, including BRCA1-A, BRCA1-B, BRCA1-C, and the BRCA1/PALB2/BRCA2 complex, and plays diverse roles in a context-dependent manner. Among the complexes, BRCA1-A is critical for BRCA1 recruitment to the sites of DNA damage. Factors comprising the BRCA1-A include RAP80, CCDC98/Abraxas, BRCC36, BRCC45, BARD1, BRCA1, and MERIT40, a RAP80-associated factor. In this review, we summarize recent findings of the factors that form the BRCA1-A complex.</P>

Chemoattractant-Mediated Rap1 Activation Requires GPCR/G Proteins

Injun Cha,Sung H. Lee,전택중 한국분자세포생물학회 2010 Molecules and cells Vol.30 No.6

Rap1 is rapidly activated upon chemoattractant stimulation and plays an important role in cell adhesion and cytoskeletal reorganization during chemotaxis. Here, we demonstrate that G-protein coupled receptors and G-proteins are essential for chemoattractant-mediated Rap1 activation in Dictyostelium. The rapid Rap1 activation upon cAMP chemoattractant stimulation was absent in cells lacking chemoattractant cAMP receptors cAR1/cAR3 or a subunit of the heterotrimeric G-protein complex Gα2. Loss of guanylyl cyclases GCA/SGC or a cGMP-binding protein GbpC exhibited no effect on Rap1 activation kinetics. These results suggest that Rap1, a key regulator for the regulation of cytoskeletal reorganization during cell movement,is activated through the G-protein coupled receptors cAR1/cAR3 and Gα2 proteins in a way independent of the cGMP signaling pathway