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IGF-I regulates tight-junction protein claudin-1 during differentiation of osteoblast-like MC3T3-E1 cells via a MAP-kinase pathway

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Abstract

Insulin-like growth factor I (IGF-I) is expressed in many tissues, including bone, and acts on the proliferation and differentiation of osteoblasts as an autocrine/paracrine regulator. Tight-junction proteins have been detected in osteoblasts, and direct cell-to-cell interactions may modulate osteoblast function with respect, for example, to gap junctions. In order to investigate the regulation of expression of tight-junction molecules and of function during bone differentiation, osteoblast-like MC3T3-E1 cells and osteocyte-like MLO-Y4 cells were treated with IGF-I. In both MC3T3-E1 cells and MLO-Y4 cells, the tight-junction molecules occludin, claudin-1, -2, and -6, and the gap-junction molecule connexin 43 (Cx43) were detected by reverse transcription with polymerase chain reaction. In MC3T3-E1 cells but not MLO-Y4 cells, mRNAs of claudin-1, -2, and -6, Cx43, and type I collagen, and proteins of claudin-1 and Cx43 were increased after treatment with IGF-I. Such treatment significantly decreased paracellular permeability in MC3T3-E1 cells. The expression of claudin-1 in MC3T3-E1 cells after IGF-I treatment was mainly upregulated via a mitogen-activated protein (MAP)-kinase pathway and, in part, modulated by a PI3-kinase pathway, whereas Cx43 expression and the mediated gap-junctional intercellular communication protein did not contribute to the upregulation. Furthermore, in MC3T3-E1 cells during wound healing, upregulation of claudin-1 was observed together with an increase of IGF-I and type I collagen. These findings suggest that the induction of tight-junction protein claudin-1 and paracellular permeability during the differentiation of osteoblast-like MC3T3-E1 cells after treatment with IGF-I is regulated via a MAP-kinase pathway, but not with respect to gap junctions.

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Acknowledgments

We thank Dr. Kenneth J. McLeod (State University of New York, Stony Brook) and Dr. Lynda F. Bonewald (University of Texas Health Science Center) for graciously providing the MC3T3-E1 and MLO-Y4 cell lines.

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Authors and Affiliations

  1. Department of Orthopaedic Surgery, Sapporo Medical University School of Medicine, S1, W17, Sapporo, 060-8556, Japan

    Naoko Hatakeyama, Kousuke Iba & Toshihiko Yamashita

  2. Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan

    Naoko Hatakeyama, Takashi Kojima, Masaki Murata, Makoto Osanai, Hideki Chiba & Norimasa Sawada

  3. Department of Neuroscience, Albert Einstein College of Medicine, Bronx, N.Y., USA

    Mia M. Thi & David C. Spray

  4. Department of Rehabilitation, Sapporo Medical University School of Medicine, Sapporo, Japan

    Sumio Ishiai

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  1. Naoko Hatakeyama
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  2. Takashi Kojima
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  3. Kousuke Iba
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Correspondence to Takashi Kojima.

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This work was supported by Grants-in-Aid from the National Project “Knowledge Cluster Initiative” (2nd stage, “Sapporo Biocluster Bio-s”), the Ministry of Education, Culture, Sports, Science, and Technology, and the Ministry of Health, Labor, and Welfare of Japan, the Akiyama Foundation, and the Japan Science and Technology Agency.

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441_2008_690_MOESM1_ESM.pdf

Fig. S. Changes of ZO-1 in MC3T3-E1 cells after treatment with IGF-I. a Western blotting for ZO-1 protein in MC3T3-E1 cells after treatment with 0.1–10 nM IGF-I. In MC3T3-E1 cells, upregulation of ZO-1 was observed from 1 nM IGF-I. The corresponding expression level of ZO-1 is shown in a bar graph. **P < 0.01 versus control. b Immunocytochemistry for ZO-1 in MC3T3-E1 cells after treatment with 1 nm IGF-I. In MC3T3-E1 cells treated with IGF-I, ZO-1-immunoreactive lines were increased compared with the control. Bar 20 mm (PDF 134 KB)

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Hatakeyama, N., Kojima, T., Iba, K. et al. IGF-I regulates tight-junction protein claudin-1 during differentiation of osteoblast-like MC3T3-E1 cells via a MAP-kinase pathway. Cell Tissue Res 334, 243–254 (2008). https://doi.org/10.1007/s00441-008-0690-9

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