Role of LRP1 in transport of CCN2 protein in chondrocytes

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Abstract

Low-density lipoprotein receptor-related protein 1 (LRP1) is known to be a receptor for signal transmission and endocytosis. We have previously reported that LRP1 regulates WNT-b-catenin and protein kinase C signaling in chondrocytes, represses the hypertrophy of chondrocytes during endochondral ossification and that LRP1 is colocalized with a ligand, CCN family member 2 (CCN2; also known as connective tissue growth factor, CTGF), which conducts endochondral ossification, in chondrocytes. However, the role of LRP1 in the endocytic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytic trafficking. Small interfering RNA (siRNA)-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding and/or incorporation was decreased in the LRP1 downregulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin, and internalizated CCN2 was colocalized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the crucial involvement of LRP1 during experimental transcytosis. Moreover, under hypoxic conditions that mimic the cartilaginous microenvironment, the level of LRP1 and the amount of transcytosed CCN2 increased, and these increases were neutralized by treatment with the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 in this tissue, which is produced by and transported by LRP1 from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which might be a crucial event that determines the distribution of CCN2 in cartilage.

Original languageEnglish
Pages (from-to)2965-2972
Number of pages8
JournalJournal of Cell Science
Volume125
Issue number12
DOIs
Publication statusPublished - Jun 15 2012

Fingerprint

Lipoprotein Receptors
Connective Tissue Growth Factor
Chondrocytes
Carrier Proteins
Proteins
Transcytosis
Osteogenesis
Low Density Lipoprotein Receptor-Related Protein-1
Catenins
Clathrin
Growth Plate
Endosomes
Endocytosis
Protein Kinase C
Hypertrophy
Small Interfering RNA
Cartilage

Keywords

  • CCN family member 2 (CCN2)
  • Chondrocytes
  • Connective tissue growth factor (CTGF)
  • Endocytosis
  • Low-density lipoprotein receptor-related protein 1 (LRP1)
  • Transcytosis

ASJC Scopus subject areas

  • Cell Biology

Cite this

@article{66e0d56ad96e4c5a99a864b4d57d9ad9,
title = "Role of LRP1 in transport of CCN2 protein in chondrocytes",
abstract = "Low-density lipoprotein receptor-related protein 1 (LRP1) is known to be a receptor for signal transmission and endocytosis. We have previously reported that LRP1 regulates WNT-b-catenin and protein kinase C signaling in chondrocytes, represses the hypertrophy of chondrocytes during endochondral ossification and that LRP1 is colocalized with a ligand, CCN family member 2 (CCN2; also known as connective tissue growth factor, CTGF), which conducts endochondral ossification, in chondrocytes. However, the role of LRP1 in the endocytic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytic trafficking. Small interfering RNA (siRNA)-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding and/or incorporation was decreased in the LRP1 downregulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin, and internalizated CCN2 was colocalized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the crucial involvement of LRP1 during experimental transcytosis. Moreover, under hypoxic conditions that mimic the cartilaginous microenvironment, the level of LRP1 and the amount of transcytosed CCN2 increased, and these increases were neutralized by treatment with the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 in this tissue, which is produced by and transported by LRP1 from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which might be a crucial event that determines the distribution of CCN2 in cartilage.",
keywords = "CCN family member 2 (CCN2), Chondrocytes, Connective tissue growth factor (CTGF), Endocytosis, Low-density lipoprotein receptor-related protein 1 (LRP1), Transcytosis",
author = "Kazumi Kawata and Satoshi Kubota and Takanori Eguchi and Eriko Aoyama and Norifumi Moritani and Seiji Kondo and Takashi Nishida and Masaharu Takigawa",
year = "2012",
month = "6",
day = "15",
doi = "10.1242/jcs.101956",
language = "English",
volume = "125",
pages = "2965--2972",
journal = "Journal of Cell Science",
issn = "0021-9533",
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TY - JOUR

T1 - Role of LRP1 in transport of CCN2 protein in chondrocytes

AU - Kawata, Kazumi

AU - Kubota, Satoshi

AU - Eguchi, Takanori

AU - Aoyama, Eriko

AU - Moritani, Norifumi

AU - Kondo, Seiji

AU - Nishida, Takashi

AU - Takigawa, Masaharu

PY - 2012/6/15

Y1 - 2012/6/15

N2 - Low-density lipoprotein receptor-related protein 1 (LRP1) is known to be a receptor for signal transmission and endocytosis. We have previously reported that LRP1 regulates WNT-b-catenin and protein kinase C signaling in chondrocytes, represses the hypertrophy of chondrocytes during endochondral ossification and that LRP1 is colocalized with a ligand, CCN family member 2 (CCN2; also known as connective tissue growth factor, CTGF), which conducts endochondral ossification, in chondrocytes. However, the role of LRP1 in the endocytic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytic trafficking. Small interfering RNA (siRNA)-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding and/or incorporation was decreased in the LRP1 downregulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin, and internalizated CCN2 was colocalized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the crucial involvement of LRP1 during experimental transcytosis. Moreover, under hypoxic conditions that mimic the cartilaginous microenvironment, the level of LRP1 and the amount of transcytosed CCN2 increased, and these increases were neutralized by treatment with the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 in this tissue, which is produced by and transported by LRP1 from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which might be a crucial event that determines the distribution of CCN2 in cartilage.

AB - Low-density lipoprotein receptor-related protein 1 (LRP1) is known to be a receptor for signal transmission and endocytosis. We have previously reported that LRP1 regulates WNT-b-catenin and protein kinase C signaling in chondrocytes, represses the hypertrophy of chondrocytes during endochondral ossification and that LRP1 is colocalized with a ligand, CCN family member 2 (CCN2; also known as connective tissue growth factor, CTGF), which conducts endochondral ossification, in chondrocytes. However, the role of LRP1 in the endocytic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytic trafficking. Small interfering RNA (siRNA)-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding and/or incorporation was decreased in the LRP1 downregulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin, and internalizated CCN2 was colocalized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the crucial involvement of LRP1 during experimental transcytosis. Moreover, under hypoxic conditions that mimic the cartilaginous microenvironment, the level of LRP1 and the amount of transcytosed CCN2 increased, and these increases were neutralized by treatment with the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 in this tissue, which is produced by and transported by LRP1 from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which might be a crucial event that determines the distribution of CCN2 in cartilage.

KW - CCN family member 2 (CCN2)

KW - Chondrocytes

KW - Connective tissue growth factor (CTGF)

KW - Endocytosis

KW - Low-density lipoprotein receptor-related protein 1 (LRP1)

KW - Transcytosis

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U2 - 10.1242/jcs.101956

DO - 10.1242/jcs.101956

M3 - Article

C2 - 22454511

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