Expression of functional gap junctions and regulation by fluid flow in osteocyte-like MLO-Y4 cells

B. Cheng, S. Zhao, J. Luo, E. Sprague, Lynda Bonewald, J. X. Jiang

Research output: Contribution to journalArticle

141 Citations (Scopus)

Abstract

Osteocytes are thought to be mechanosensory cells that respond to mechanical stress by sending signals to other bone cells to initiate bone remodeling. An osteocyte-like cell line MLO-Y4 provides a model system to examine whether gap junctions participate in the regulation of osteocyte function and signaling by mechanical stress. In this study, we show that MLO-Y4 cells are coupled and that gap junction channels mediate this coupling. Biochemical analyses show that connexin 43 (Cx43) is a major gap junction protein expressed in MLO-Y4 cells and approximately 5% of Cx43 protein is phosphorylated. MLO-Y4 cells were exposed to mechanical stress using a parallel plate flow chamber to model bone fluid flow shear stress. Fluid flow increased significantly the length of the dendritic processes, a morphological characteristic of osteocytes. A redistribution of the gap junction protein, Cx43 also was observed from a location circling the nucleus to punctate spots in the cytoplasm and in the dendritic processes. "Scrape-loading" dye transfer analyses showed that fluid flow increased intercellular coupling and increased the number of cells coupled immediately after fluid flow treatment, in direct proportion to shear stress magnitude. Although intercellular coupling continued to increase, stimulation of Cx43 protein expression during the poststress period was found to be biphasic. Cx43 protein was elevated 30 minutes after application of stress but decreased at 24 h poststress. Pulsating fluid flow had a similar stimulatory effect as steady fluid flow on gap junctions. However, this stimulatory effect in osteocyte-like cells was not observed in osteoblast-like 2T3 cells. Together, these results show that fluid flow has stimulatory effects on osteocyte-like MLO-Y4 cells with early effects on cellular morphology, opening of gap junctions, and redistribution of Cx43 protein and delayed effects on Cx43 protein expression. The high expression of Cx43 and its location in the cytoplasm also suggest that Cx43 may have unknown functions in addition to forming gap junctions. These studies indicate that gap junctions may serve as channels for signals generated by osteocytes in response to mechanical loading.

Original languageEnglish (US)
Pages (from-to)249-259
Number of pages11
JournalJournal of Bone and Mineral Research
Volume16
Issue number2
StatePublished - 2001
Externally publishedYes

Fingerprint

Osteocytes
Connexin 43
Gap Junctions
Mechanical Stress
Connexins
Proteins
Cytoplasm
Bone and Bones
Pulsatile Flow
Bone Remodeling
Osteoblasts
Coloring Agents
Cell Count
Cell Line

Keywords

  • Connexin 43
  • Fluid flow
  • Gap junctions
  • MLO-Y4 cells
  • Osteocytes

ASJC Scopus subject areas

  • Surgery

Cite this

Expression of functional gap junctions and regulation by fluid flow in osteocyte-like MLO-Y4 cells. / Cheng, B.; Zhao, S.; Luo, J.; Sprague, E.; Bonewald, Lynda; Jiang, J. X.

In: Journal of Bone and Mineral Research, Vol. 16, No. 2, 2001, p. 249-259.

Research output: Contribution to journalArticle

Cheng, B. ; Zhao, S. ; Luo, J. ; Sprague, E. ; Bonewald, Lynda ; Jiang, J. X. / Expression of functional gap junctions and regulation by fluid flow in osteocyte-like MLO-Y4 cells. In: Journal of Bone and Mineral Research. 2001 ; Vol. 16, No. 2. pp. 249-259.
@article{7b7da39615c544d88fed7f9c8cf21195,
title = "Expression of functional gap junctions and regulation by fluid flow in osteocyte-like MLO-Y4 cells",
abstract = "Osteocytes are thought to be mechanosensory cells that respond to mechanical stress by sending signals to other bone cells to initiate bone remodeling. An osteocyte-like cell line MLO-Y4 provides a model system to examine whether gap junctions participate in the regulation of osteocyte function and signaling by mechanical stress. In this study, we show that MLO-Y4 cells are coupled and that gap junction channels mediate this coupling. Biochemical analyses show that connexin 43 (Cx43) is a major gap junction protein expressed in MLO-Y4 cells and approximately 5{\%} of Cx43 protein is phosphorylated. MLO-Y4 cells were exposed to mechanical stress using a parallel plate flow chamber to model bone fluid flow shear stress. Fluid flow increased significantly the length of the dendritic processes, a morphological characteristic of osteocytes. A redistribution of the gap junction protein, Cx43 also was observed from a location circling the nucleus to punctate spots in the cytoplasm and in the dendritic processes. {"}Scrape-loading{"} dye transfer analyses showed that fluid flow increased intercellular coupling and increased the number of cells coupled immediately after fluid flow treatment, in direct proportion to shear stress magnitude. Although intercellular coupling continued to increase, stimulation of Cx43 protein expression during the poststress period was found to be biphasic. Cx43 protein was elevated 30 minutes after application of stress but decreased at 24 h poststress. Pulsating fluid flow had a similar stimulatory effect as steady fluid flow on gap junctions. However, this stimulatory effect in osteocyte-like cells was not observed in osteoblast-like 2T3 cells. Together, these results show that fluid flow has stimulatory effects on osteocyte-like MLO-Y4 cells with early effects on cellular morphology, opening of gap junctions, and redistribution of Cx43 protein and delayed effects on Cx43 protein expression. The high expression of Cx43 and its location in the cytoplasm also suggest that Cx43 may have unknown functions in addition to forming gap junctions. These studies indicate that gap junctions may serve as channels for signals generated by osteocytes in response to mechanical loading.",
keywords = "Connexin 43, Fluid flow, Gap junctions, MLO-Y4 cells, Osteocytes",
author = "B. Cheng and S. Zhao and J. Luo and E. Sprague and Lynda Bonewald and Jiang, {J. X.}",
year = "2001",
language = "English (US)",
volume = "16",
pages = "249--259",
journal = "Journal of Bone and Mineral Research",
issn = "0884-0431",
publisher = "Wiley-Blackwell",
number = "2",

}

TY - JOUR

T1 - Expression of functional gap junctions and regulation by fluid flow in osteocyte-like MLO-Y4 cells

AU - Cheng, B.

AU - Zhao, S.

AU - Luo, J.

AU - Sprague, E.

AU - Bonewald, Lynda

AU - Jiang, J. X.

PY - 2001

Y1 - 2001

N2 - Osteocytes are thought to be mechanosensory cells that respond to mechanical stress by sending signals to other bone cells to initiate bone remodeling. An osteocyte-like cell line MLO-Y4 provides a model system to examine whether gap junctions participate in the regulation of osteocyte function and signaling by mechanical stress. In this study, we show that MLO-Y4 cells are coupled and that gap junction channels mediate this coupling. Biochemical analyses show that connexin 43 (Cx43) is a major gap junction protein expressed in MLO-Y4 cells and approximately 5% of Cx43 protein is phosphorylated. MLO-Y4 cells were exposed to mechanical stress using a parallel plate flow chamber to model bone fluid flow shear stress. Fluid flow increased significantly the length of the dendritic processes, a morphological characteristic of osteocytes. A redistribution of the gap junction protein, Cx43 also was observed from a location circling the nucleus to punctate spots in the cytoplasm and in the dendritic processes. "Scrape-loading" dye transfer analyses showed that fluid flow increased intercellular coupling and increased the number of cells coupled immediately after fluid flow treatment, in direct proportion to shear stress magnitude. Although intercellular coupling continued to increase, stimulation of Cx43 protein expression during the poststress period was found to be biphasic. Cx43 protein was elevated 30 minutes after application of stress but decreased at 24 h poststress. Pulsating fluid flow had a similar stimulatory effect as steady fluid flow on gap junctions. However, this stimulatory effect in osteocyte-like cells was not observed in osteoblast-like 2T3 cells. Together, these results show that fluid flow has stimulatory effects on osteocyte-like MLO-Y4 cells with early effects on cellular morphology, opening of gap junctions, and redistribution of Cx43 protein and delayed effects on Cx43 protein expression. The high expression of Cx43 and its location in the cytoplasm also suggest that Cx43 may have unknown functions in addition to forming gap junctions. These studies indicate that gap junctions may serve as channels for signals generated by osteocytes in response to mechanical loading.

AB - Osteocytes are thought to be mechanosensory cells that respond to mechanical stress by sending signals to other bone cells to initiate bone remodeling. An osteocyte-like cell line MLO-Y4 provides a model system to examine whether gap junctions participate in the regulation of osteocyte function and signaling by mechanical stress. In this study, we show that MLO-Y4 cells are coupled and that gap junction channels mediate this coupling. Biochemical analyses show that connexin 43 (Cx43) is a major gap junction protein expressed in MLO-Y4 cells and approximately 5% of Cx43 protein is phosphorylated. MLO-Y4 cells were exposed to mechanical stress using a parallel plate flow chamber to model bone fluid flow shear stress. Fluid flow increased significantly the length of the dendritic processes, a morphological characteristic of osteocytes. A redistribution of the gap junction protein, Cx43 also was observed from a location circling the nucleus to punctate spots in the cytoplasm and in the dendritic processes. "Scrape-loading" dye transfer analyses showed that fluid flow increased intercellular coupling and increased the number of cells coupled immediately after fluid flow treatment, in direct proportion to shear stress magnitude. Although intercellular coupling continued to increase, stimulation of Cx43 protein expression during the poststress period was found to be biphasic. Cx43 protein was elevated 30 minutes after application of stress but decreased at 24 h poststress. Pulsating fluid flow had a similar stimulatory effect as steady fluid flow on gap junctions. However, this stimulatory effect in osteocyte-like cells was not observed in osteoblast-like 2T3 cells. Together, these results show that fluid flow has stimulatory effects on osteocyte-like MLO-Y4 cells with early effects on cellular morphology, opening of gap junctions, and redistribution of Cx43 protein and delayed effects on Cx43 protein expression. The high expression of Cx43 and its location in the cytoplasm also suggest that Cx43 may have unknown functions in addition to forming gap junctions. These studies indicate that gap junctions may serve as channels for signals generated by osteocytes in response to mechanical loading.

KW - Connexin 43

KW - Fluid flow

KW - Gap junctions

KW - MLO-Y4 cells

KW - Osteocytes

UR - http://www.scopus.com/inward/record.url?scp=0035141749&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035141749&partnerID=8YFLogxK

M3 - Article

VL - 16

SP - 249

EP - 259

JO - Journal of Bone and Mineral Research

JF - Journal of Bone and Mineral Research

SN - 0884-0431

IS - 2

ER -