Enhanced gap junction intercellular communication inhibits catabolic and pro-inflammatory responses in tenocytes against heat stress

Abstract

Elevation of tendon core temperature during severe activity is well known. However, its effects on tenocyte function have not been studied in detail. The present study tested a hypothesis that heat stimulation upregulates tenocyte catabolism, which can be modulated by the inhibition or the enhancement of gap junction intercellular communication (GJIC). Tenocytes isolated from rabbit Achilles tendons were subjected to heat stimulation at 37 °C, 41 °C or 43 °C for 30 min, and changes in cell viability, gene expressions and GJIC were examined. It was found that GJIC exhibited no changes by the stimulation even at 43 °C, but cell viability was decreased and catabolic and proinflammatory gene expressions were upregulated. Inhibition of GJIC demonstrated further upregulated catabolic and proinflammatory gene expressions. In contrast, enhanced GJIC, resulting from forced upregulation of connexin 43 gene, counteracted the heat-induced upregulation of catabolic and proinflammatory genes. These findings suggest that the temperature rise in tendon core could upregulate catabolic and proinflammatory activities, potentially leading to the onset of tendinopathy, and such upregulations could be suppressed by the enhancement of GJIC. Therefore, to prevent tendon injury at an early stage from becoming chronic injury, tendon core temperature and GJIC could be targets for post-activity treatments.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Abbreviations

Casp:

Caspase

Col1a2:

Type I collagen alpha 2 chain

Cx:

Connexin

FLIP:

Fluorescence loss in photobleaching

Gapdh:

Glyceraldehyde 3-phosphate dehydrogenase

GJIC:

Gap junction intercellular communication

Hsp:

Heat shock protein

IL:

Interluekin

MMP:

Matrix metalloproteinase

PDMS:

Polydimethylsiloxane

TRPV1:

Transient receptor potential vanilloid-1

18αGA :

18α glycyrrhetinic acid

References

  1. Antanavičiute I, Mildažiene V, Stankevičius E et al (2014) Hyperthermia differently affects connexin43 expression and gap junction permeability in skeletal myoblasts and Hela cells. Mediat Inflamm 2014:748290. doi:10.1155/2014/748290

    Google Scholar 

  2. Banes AJ, Weinhold P, Yang X et al (1999) Gap junctions regulate responses of tendon cells ex vivo to mechanical loading. Clin Orthop Relat Res 367S:S356–S370

  3. Birch HL, Wilson AM, Goodship AE (1997) The effect of exercise-induced localised hyperthermia on tendon cell survival. J Exp Biol 200:1703–1708

    CAS  PubMed  Google Scholar 

  4. Bischof JC, Padanilam J, Holmes WH et al (1995) Dynamics of cell membrane permeability changes at supraphysiological temperatures. Biophys J 68:2608–2614. doi:10.1016/S0006-3495(95)80445-5

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Blanc EM, Bruce-Keller AJ, Mattson MP (1998) Astrocytic gap junctional communication decreases neuronal vulnerability to oxidative stress-induced disruption of Ca2+ homeostasis and cell death. J Neurochem 70:958–970. doi:10.1046/j.1471-4159.1998.70030958.x

    CAS  Article  PubMed  Google Scholar 

  6. Brosnan CF, Scemes E, Spray DC (2001) Cytokine regulation of gap junction connectivity. Am J Pathol 158:1565–1569. doi:10.1016/S0002-9440(10)64110-7

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Bukauskas FF, Verselis VK (2004) Gap junction channel gating. Biochim Biophys Acta Biomembr 1662:42–60. doi:10.1016/j.bbamem.2004.01.008

    CAS  Article  Google Scholar 

  8. Caterina MJ, Schumacher MA, Tominaga M et al (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389:816–824. doi:10.1038/39807

    CAS  Article  PubMed  Google Scholar 

  9. Egerbacher M, Arnoczky SP, Caballero O et al (2008) Loss of homeostatic tension induces apoptosis in tendon cells: an in vitro study. Clin Orthop Relat Res 466:1562–1568. doi:10.1007/s11999-008-0274-8

    Article  PubMed  PubMed Central  Google Scholar 

  10. Farris DJ, Trewartha G, Polly McGuigan M (2011) Could intra-tendinous hyperthermia during running explain chronic injury of the human Achilles tendon? J Biomech 44:822–826. doi:10.1016/j.jbiomech.2010.12.015

    Article  PubMed  Google Scholar 

  11. García-Dorado D, Rodríguez-Sinovas A, Ruiz-Meana M (2004) Gap junction-mediated spread of cell injury and death during myocardial ischemia-reperfusion. Cardiovasc Res 61:386–401. doi:10.1016/j.cardiores.2003.11.039

    Article  PubMed  Google Scholar 

  12. Hamada N, Kodama S, Suzuki K, Watanabe M (2003) Gap junctional intercellular communication and cellular response to heat stress. Carcinogenesis 24:1723–1728. doi:10.1093/carcin/bgg135

    CAS  Article  PubMed  Google Scholar 

  13. Hayes P, Meadows HJ, Gunthorpe MJ et al (2000) Cloning and functional expression of a human orthologue of rat vanilloid receptor-1. Pain 88:205–215. doi:10.1016/S0304-3959(00)00353-5

    CAS  Article  PubMed  Google Scholar 

  14. Hosaka Y, Ozoe S, Kirisawa R et al (2006) Effect of heat on synthesis of gelatinases and pro-inflammatory cytokines in equine tendinocytes. Biomed Res 27:233–241. doi:10.2220/biomedres.27.233

    CAS  Article  PubMed  Google Scholar 

  15. Lavagnino M, Arnoczky SP (2005) In vitro alterations in cytoskeletal tensional homeostasis control gene expression in tendon cells. J Orthop Res 23:1211–1218. doi:10.1016/j.orthres.2005.04.001

    CAS  Article  PubMed  Google Scholar 

  16. Lavagnino M, Arnoczky SP, Tian T, Vaupel Z (2003) Effect of amplitude and frequency of cyclic tensile strain on the inhibition of MMP-1 mRNA expression in tendon cells: an in vitro study. Connect Tissue Res 44:181–187. doi:10.1080/03008200390215881

    CAS  Article  PubMed  Google Scholar 

  17. Lavagnino M, Arnoczky SP, Egerbacher M et al (2006) Isolated fibrillar damage in tendons stimulates local collagenase mRNA expression and protein synthesis. J Biomech 39:2355–2362. doi:10.1016/j.jbiomech.2005.08.008

    Article  PubMed  Google Scholar 

  18. Lee YM, Li WH, Kim YK et al (2008) Heat-induced MMP-1 expression is mediated by TRPV1 through PKCa signaling in HaCaT cells. Exp Dermatol 17:864–870. doi:10.1111/j.1600-0625.2008.00738.x

    CAS  Article  PubMed  Google Scholar 

  19. Legerlotz K, Jones GC, Screen HRC, Riley GP (2013) Cyclic loading of tendon fascicles using a novel fatigue loading system increases interleukin-6 expression by tenocytes. Scand J Med Sci Sports 23:31–37. doi:10.1111/j.1600-0838.2011.01410.x

    CAS  Article  PubMed  Google Scholar 

  20. Levin M (2007) Gap junctional communication in morphogenesis. Prog Biophys Mol Biol 94:186–206. doi:10.1016/j.pbiomolbio.2007.03.005

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Li WH, Lee YM, Kim JY et al (2007) Transient receptor potential vanilloid-1 mediates heat-shock-induced matrix metalloproteinase-1 expression in human epidermal keratinocytes. J Invest Dermatol 127:2328–2335. doi:10.1038/sj.jid.5700880

    CAS  Article  PubMed  Google Scholar 

  22. Lin JH-C, Yang J, Liu S et al (2003) Connexin mediates gap junction-independent resistance to cellular injury. J Neurosci 23:430–441

    CAS  PubMed  Google Scholar 

  23. Maeda E, Ohashi T (2015) Mechano-regulation of gap junction communications between tendon cells is dependent on the magnitude of tensile strain. Biochem Biophys Res Commun 465:281–286. doi:10.1016/j.bbrc.2015.08.021

    CAS  Article  PubMed  Google Scholar 

  24. Maeda E, Ye S, Wang W et al (2012) Gap junction permeability between tenocytes within tendon fascicles is suppressed by tensile loading. Biomech Model Mechanobiol 11:439–447

    Article  PubMed  Google Scholar 

  25. Maeda E, Hagiwara Y, Wang JHC, Ohashi T (2013) A new experimental system for simultaneous application of cyclic tensile strain and fluid shear stress to tenocytes in vitro. Biomed Microdevices 15:1067–1075. doi:10.1007/s10544-013-9798-0

    Article  PubMed  Google Scholar 

  26. Maeda E, Pian H, Ohashi T (2017) Temporal regulation of gap junctional communication between tenocytes subjected to static tensile strain with physiological and non-physiological amplitudes. Biochem Biophys Res Commun 482:1170–1175. doi:10.1016/j.bbrc.2016.12.007

    CAS  Article  PubMed  Google Scholar 

  27. Nagata K, Saga S, Yamada KM (1986) A major collagen-binding protein of chick embryo fibroblasts is a novel heat shock protein. J Cell Biol 103:223–229

    CAS  Article  PubMed  Google Scholar 

  28. Ning S, Hahn GM (1994) Formation of tight junctions and desmosomes protects MDCK cells against hyperthermic killing. J Cell Physiol 160:249–254. doi:10.1002/jcp.1041600206

    CAS  Article  PubMed  Google Scholar 

  29. Patterson-Kane JC, Becker DL, Rich T (2012) The pathogenesis of tendon Microdamage in athletes: the horse as a natural model for basic cellular research. J Comp Pathol 147:227–247. doi:10.1016/j.jcpa.2012.05.010

    CAS  Article  PubMed  Google Scholar 

  30. Pitts JD (1998) The discovery of metabolic co-operation. BioEssays 20:1047–1051. doi:10.1002/(SICI)1521-1878(199812)20:12<1047::AID-BIES11>3.0.CO;2-0

    CAS  Article  PubMed  Google Scholar 

  31. Qi J, Chi L, Bynum D, Banes AJ (2011) Gap junctions in IL-1beta-mediated cell survival response to strain. J Appl Physiol 110:1425–1431. doi:10.1152/japplphysiol.00477.2010

    CAS  Article  PubMed  Google Scholar 

  32. Scott A, Khan KM, Heer J et al (2005) High strain mechanical loading rapidly induces tendon apoptosis: an ex vivo rat tibialis anterior model. Br J Sports Med 39:e25. doi:10.1136/bjsm.2004.015164

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Son GY, Hong JH, Chang I, Shin DM (2015) Induction of IL-6 and IL-8 by activation of thermosensitive TRP channels in human PDL cells. Arch Oral Biol 60:526–532. doi:10.1016/j.archoralbio.2014.12.014

    CAS  Article  PubMed  Google Scholar 

  34. Sun HB, Li Y, Fung DT et al (2008) Coordinate regulation of IL-1b and MMP-13 in rat tendons following subrupture fatigue damage. Clin Orthop Relat Res 466:1555–1561. doi:10.1007/s11999-008-0278-4

    Article  PubMed  PubMed Central  Google Scholar 

  35. Waggett AD, Benjamin M, Ralphs JR (2006) Connexin 32 and 43 gap junctions differentially modulate tenocyte response to cyclic mechanical load. Eur J Cell Biol 85:1145–1154. doi:10.1016/j.ejcb.2006.06.002

    CAS  Article  PubMed  Google Scholar 

  36. Wall ME, Banes AJ (2005) Early responses to mechanical load in tendon: role for calcium signaling, gap junctions and intercellular communication. J Musculoskelet Neuronal Interact 5:70–84

    CAS  PubMed  Google Scholar 

  37. Wang JHC (2006) Mechanobiology of tendon. J Biomech 39:1563–1582. doi:10.1016/j.jbiomech.2005.05.011

    Article  PubMed  Google Scholar 

  38. Wilson AM, Goodship AE (1994) Exercise-induced hyperthermia as a possible mechanism for tendon degeneration. J Biomech 27:899–905. doi:10.1016/0021-9290(94)90262-3

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

EM thanks Professor Takeo Matsumoto in Nagoya University for generous supports. The present study was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grants 25702022 and 16 K01346.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Eijiro Maeda.

Ethics declarations

Conflict of interests

The authors declare no conflicts of interest.

Electronic supplementary material

ESM 1

(DOCX 72 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Maeda, E., Kimura, S., Yamada, M. et al. Enhanced gap junction intercellular communication inhibits catabolic and pro-inflammatory responses in tenocytes against heat stress. J. Cell Commun. Signal. 11, 369–380 (2017). https://doi.org/10.1007/s12079-017-0397-3

Download citation

Keywords

  • FLIP
  • Gap junction
  • Heat stimulation
  • Intercellular communication
  • Tendinopathy
  • Tenocytes