Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes
Abstract
:1. Introduction
2. Results
2.1. The Gene Expression of Rab14 and Ccn2 in Mouse Tissues
2.2. Rab14 Interacts with the IGFBP-like Domain of CCN2
2.3. Association of Intracellular CCN2 with Rab14 on Vesicles During Their Transport in Cells
2.4. Induction of ER and Golgi Stress by the Suppression of RAB14 or CCN2
2.5. Reduction in Aggrecan Accumulation in HCS-2/8 Cells by Rab14DN Overexpression
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Total RNA Extraction, Reverse-Transcription, and qRT-PCR
4.3. The Analysis of CCN2 Domain Interacting to Rab14
4.4. In Situ Proximity Ligation Assay (PLA)
4.5. Expression Vectors
4.6. Western Blotting
4.7. Fluorescence Imaging
4.8. Indirect Immunofluorescence and Fluorescence Deconvolution Microscopy
4.9. Small Interfering RNA (siRNA) Experiments
4.10. Toluidine Blue and Alcian Blue Staining
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- Perbal, B.; Takigawa, M. CCN Proteins: A New Family of Cell Growth and Differentiation Regulators; Imperial College Press: London, UK, 2005. [Google Scholar]
- Perbal, B.; Tweedie, S.; Bruford, E. The official unified nomenclature adopted by the HGNC calls for the use of the acronyms, CCN1-6, and discontinuation in the use of CYR61, CTGF, NOV and WISP 1–3 respectively. J. Cell Commun. Signal. 2018, 12, 625–629. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Takigawa, M. CCN2: A master regulator of the genesis of bone and cartilage. J. Cell Commun. Signal. 2013, 7, 191–201. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Takigawa, M. CTGF/Hcs24 as a multifunctional growth factor for fibroblasts, chondrocytes and vascular endothelial cells. Drug News Perspect. 2003, 16, 11–21. [Google Scholar] [CrossRef]
- Takigawa, M. The CCN proteins. Methods in Molecular Biology. 2017, 1489, 1–8. [Google Scholar]
- Takigawa, M.; Nakanishi, T.; Kubota, S.; Nishida, T. Role of CTGF/HCS24/ecogenin in skeletal growth control. J. Cell. Physiol. 2003, 194, 256–266. [Google Scholar] [CrossRef]
- Ivkovic, S.; Yoon, B.S.; Popoff, S.N.; Safadi, F.F.; Libuda, D.E.; Stephenson, R.C.; Daluiski, A.; Lyons, K.M. Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 2003, 130, 2779–2791. [Google Scholar] [CrossRef] [Green Version]
- Hoshijima, M.; Hattori, T.; Inoue, M.; Araki, D.; Hanagata, H.; Miyauchi, A.; Takigawa, M. CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin α5β1. FEBS Lett. 2006, 580, 1376–1382. [Google Scholar] [CrossRef] [Green Version]
- Aoyama, E.; Hattori, T.; Hoshijima, M.; Araki, D.; Nishida, T.; Kubota, S.; Takigawa, M. N-terminal domains of CCN family 2/connective tissue growth factor bind to aggrecan. Biochem. J. 2009, 420, 413–420. [Google Scholar] [CrossRef] [Green Version]
- Hoshijima, M.; Hattori, T.; Aoyama, E.; Nishida, T.; Yamashiro, T.; Takigawa, M. Roles of heterotypic CCN2/CTGF-CCN3/NOV and homotypic CCN2-CCN2 interactions in expression of the differentiated phenotype of chondrocytes. FEBS J. 2012, 279, 3584–3597. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Sabbah, M.; Prunier, C.; Ferrand, N.; Megalophonos, V.; Lambein, K.; De Wever, O.; Nazaret, N.; Lachuer, J.; Dumont, S.; Redeuilh, G. CCN5, a novel transcriptional repressor of the transforming growth factor β signaling pathway. Mol. Cell Biol. 2011, 31, 1459–1469. [Google Scholar] [CrossRef] [Green Version]
- Takigawa, M.; Tajima, K.; Pan, H.O.; Enomoto, M.; Kinoshita, A.; Suzuki, F.; Takano, Y.; Mori, Y. Establishment of a clonal human chondrosarcoma cell line with cartilage phenotypes. Cancer Res. 1989, 49, 3996–4002. [Google Scholar] [PubMed]
- Zerial, M.; McBride, H. Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2001, 2, 107–117. [Google Scholar] [CrossRef]
- Langemeyer, L.; Fröhlich, F.; Ungermann, C. Rab GTPase Function in Endosome and Lysosome Biogenesis. Trends Cell Biol. 2018, 28, 957–970. [Google Scholar] [CrossRef] [PubMed]
- Junutula, J.R.; De Maziére, A.M.; Peden, A.A.; Ervin, K.E.; Advani, R.J.; van Dijk, S.M.; Klumperman, J.; Scheller, R.H. Rab14 is involved in membrane trafficking between the Golgi complex and endosomes. Mol. Biol. Cell 2004, 15, 2218–2229. [Google Scholar] [CrossRef] [PubMed]
- Ueno, H.; Huang, X.; Tanaka, Y.; Hirokawa, N. KIF16B/Rab14 molecular motor complex is critical for early embryonic development by transporting FGF receptor. Dev. Cell 2011, 20, 60–71. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yang, L.; Clinton, J.M.; Blackburn, M.L.; Zhang, Q.; Zou, J.; Zielinska-Kwiatkowska, A.; Tang, B.L.; Chansky, H.A. Rab23 regulates differentiation of ATDC5 chondroprogenitor cells. J. Biol. Chem. 2008, 283, 10649–10657. [Google Scholar] [CrossRef] [Green Version]
- Eguchi, T.; Kubota, S.; Kondo, S.; Shimo, T.; Hattori, T.; Nakanishi, T.; Kuboki, T.; Yatani, H.; Takigawa, M. Regulatory mechanism of human connective tissue growth factor (CTGF/Hcs24) gene expression in a human chondrocytic cell line, HCS-2/8. J. Biochem. 2001, 130, 79–87. [Google Scholar] [CrossRef]
- Pfeffer, S.R. Rab GTPases: Master regulators that establish the secretory and endocytic pathways. Mol. Biol. Cell 2017, 28, 712–715. [Google Scholar] [CrossRef]
- Itoh, T.; Fujita, N.; Kanno, E.; Yamamoto, A.; Yoshimori, T.; Fukuda, M. Golgi-resident small GTPase Rab33B interacts with Atg16L and modulates autophagosome formation. Mol. Biol. Cell 2008, 19, 2916–2925. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Munafo, D.B.; Johnson, J.L.; Ellis, B.A.; Rutschmann, S.; Beutler, B.; Catz, S.D. Rab27a is a key component of the secretory machinery of azurophilic granules in granulocytes. Biochem. J. 2007, 402, 229–239. [Google Scholar] [CrossRef] [Green Version]
- Gomi, H.; Mizutani, S.; Kasai, K.; Itohara, S.; Izumi, T. Granuphilin molecularly docks insulin granules to the fusion machinery. J. Cell Biol. 2005, 171, 99–109. [Google Scholar] [CrossRef] [PubMed]
- Fukuda, M. Versatile role of Rab27 in membrane trafficking: Focus on the Rab27 effector families. J. Biochem. 2005, 137, 9–16. [Google Scholar] [CrossRef] [PubMed]
- Fukuda, M.; Kanno, E.; Ishibashi, K.; Itoh, T. Large scale screening for novel Rab effectors reveals unexpected broad Rab binding specificity. Mol. Cell. Proteomics 2008, 7, 1031–1042. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lu, R.; Dalgalan, D.; Mandell, E.K.; Parker, S.S.; Ghosh, S.; Wilson, J.M. PKCι interacts with Rab14 and modulates epithelial barrier function through regulation of claudin-2 levels. Mol. Biol. Cell 2015, 26, 1523–1531. [Google Scholar] [CrossRef] [PubMed]
- Abreu, J.G.; Ketpura, N.I.; Reversade, B.; De Robertis, E.M. Connective tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-β. Nat. Cell Biol. 2002, 4, 599–604. [Google Scholar] [CrossRef] [Green Version]
- Jun, J.I.; Lau, L.F. Taking aim at the extracellular matrix: CCN proteins as emerging therapeutic targets. Nat Rev Drug Discov. 2011, 10, 945–963. [Google Scholar] [CrossRef] [Green Version]
- Kubota, S.; Takigawa, M. The CCN family acting throughout the body: Recent research developments. Biomol. Concepts 2013, 4, 477–494. [Google Scholar] [CrossRef]
- Cooper, A.A.; Gitler, A.D.; Cashikar, A.; Haynes, C.M.; Hill, K.J.; Bhullar, B.; Liu, K.; Xu, K.; Strathearn, K.E.; Liu, F.; et al. Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues. neuron loss in Parkinson’s models. Science 2006, 313, 324–328. [Google Scholar] [CrossRef] [Green Version]
- Maeda-Uematsu, A.; Kubota, S.; Kawaki, H.; Kawata, K.; Miyake, Y.; Hattori, T.; Nishida, T.; Moritani, N.; Lyons, K.M.; Iida, S.; et al. CCN2 as a novel molecule supporting energy metabolism of chondrocytes. J. Cell Biochem. 2014, 115, 854–865. [Google Scholar] [CrossRef] [Green Version]
- Kamatsuki, Y.; Aoyama, E.; Furumatsu, T.; Miyazawa, S.; Maehara, A.; Yamanaka, N.; Nishida, T.; Kubota, S.; Ozaki, T.; Takigawa, M. Possible reparative effect of low-intensity pulsed ultrasound (LIPUS) on injured meniscus. J. Cell Commun. Signal. 2019, 13, 193–207. [Google Scholar] [CrossRef]
- Nishida, T.; Kawaki, H.; Baxter, R.M.; Deyoung, R.A.; Takigawa, M.; Lyons, K.M. CCN2 (Connective Tissue Growth Factor) is essential for extracellular matrix production and integrin signaling in chondrocytes. J. Cell Commun. Signal. 2007, 1, 45–58. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hattori, T.; Eberspaecher, H.; Lu, J.; Zhang, R.; Nishida, T.; Kahyo, T.; Yasuda, H.; de Crombrugghe, B. Interactions between PIAS proteins and SOX9 result in an increase in the cellular concentrations of SOX9. J. Biol. Chem. 2006, 281, 14417–14428. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hattori, T.; von der Mark, K.; Kawaki, H.; Yutani, Y.; Kubota, S.; Nakanishi, T.; Eberspaecher, H.; de Crombrugghe, B.; Takigawa, M. Downregulation of rheumatoid arthritis-related antigen RA-A47 (HSP47/colligin-2) in chondrocytic cell lines induces apoptosis and cell-surface expression of RA-A47 in association with CD9. J. Cell. Physiol. 2005, 202, 191–204. [Google Scholar] [CrossRef] [PubMed]
- Hoshijima, M.; Hattori, T.; Takigawa, M. Protocols for screening for binding partners of CCN proteins: Yeast two-hybrid system. Methods Mol. Biol. 2017, 1489, 145–154. [Google Scholar] [PubMed]
- Kobayashi, D.; Endo, M.; Ochi, H.; Hojo, H.; Miyasaka, M.; Hayasaka, H. Regulation of CCR7-dependent cell migration through CCR7 homodimer formation. Sci. Rep. 2017, 7, 8536. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hattori, T.; Müller, C.; Gebhard, S.; Bauer, E.; Pausch, F.; Schlund, B.; Bösl, M.R.; Hess, A.; Surmann-Schmitt, C.; von der Mark, H.; et al. SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. Development 2010, 137, 901–911. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Gene | Forward | Reverse |
---|---|---|
Ccn2 | ccacccgagttaccaatgac | gtgcagccagaaagctca |
Rab14 | gcagatttgggatacagcaggg | cagtgtttggattggtgagattcc |
Gapdh * | caatgaccccttcattgacc | gacaagcttcccgttctcag |
Gene | Forward | Reverse |
---|---|---|
CCN2 | gcaggctagagaagcagagc | atgtcttcatgctggtgcag |
RAB14 | agatttgggatacggcaggac | cagtatttggattggtgagattcc |
ACAN | gaggagagaactggagaag | gccgatagtggaatacaac |
BIP | gtttgctgaggaagacaaaaagctc | cacttccatagagtttgctgataat |
CHOP | gtccagctgggagctggaag | ctgactggaatctggagag |
SIAT4A | gataagatcctgatctaccacc | ttgttctcccagtagtggtg |
18S rRNA | gcgaattcctgccagtagcatatgctg | ggaagcttagaggagcgagcgaccaaagg |
CCN2 Fragment | Forward | Reverse |
---|---|---|
Full-length | atccgaattccagaactgcagcgggccgtgccggtgcccg | atacggatccctcatgccatgtctccgtacatcttcctgt |
VWC-TSP1-CT | atccgaattcgtgtgcaccgccaaagatggtgctccctgc | atacggatccctcatgccatgtctccgtacatcttcctgt |
TSP1-CT | atccgaattcactatgattagagccaactgcctggtccaga | atacggatccctcatgccatgtctccgtacatcttcctgt |
CT | atccgaattcaacattaagaagggcaaaaagtgcatccgt | atacggatccctcatgccatgtctccgtacatcttcctgt |
IGFBP | atccgaattccagaactgcagcgggccgtgccggtgcccg | atacggatccgagcaccatctttggcggtgcacacgccga |
VWC | atccgaattcgtgtgcaccgccaaagatggtgctccctgc | atacggatccagttggctctaatcatagttgggtctgggc |
TSP1 | atccgaattcactatgattagagccaactgcctggtccaga | atacggatccggatgcactttttgcccttcttaatgttct |
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Hoshijima, M.; Hattori, T.; Aoyama, E.; Nishida, T.; Kubota, S.; Kamioka, H.; Takigawa, M. Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes. Int. J. Mol. Sci. 2020, 21, 2769. https://doi.org/10.3390/ijms21082769
Hoshijima M, Hattori T, Aoyama E, Nishida T, Kubota S, Kamioka H, Takigawa M. Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes. International Journal of Molecular Sciences. 2020; 21(8):2769. https://doi.org/10.3390/ijms21082769
Chicago/Turabian StyleHoshijima, Mitsuhiro, Takako Hattori, Eriko Aoyama, Takashi Nishida, Satoshi Kubota, Hiroshi Kamioka, and Masaharu Takigawa. 2020. "Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes" International Journal of Molecular Sciences 21, no. 8: 2769. https://doi.org/10.3390/ijms21082769
APA StyleHoshijima, M., Hattori, T., Aoyama, E., Nishida, T., Kubota, S., Kamioka, H., & Takigawa, M. (2020). Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes. International Journal of Molecular Sciences, 21(8), 2769. https://doi.org/10.3390/ijms21082769