1. HEAT SHOCK PROTEINS IN BALNEOTHERAPY
Prof Dr Nergis Erdoğan
Turkish Hungarian Balneotherapy Congress Balçova İzmir 2006

2. Heat Shock (Stress) Proteins
Why heat shock proteins interest us?
What Are they?
What are the functions of HSPs?
The HSP70 family
Thermotolerance
HSP70 functions associated with stress tolerance
Factors and conditions that modulate hsp70 expression
Summary and future directions
Turkish Hungarian Balneotherapy Congress Balçova İzmir 2006

3. Why do heat shock proteins interest us?
Thermomineral or hyperthermal water baths take an important place in balneotherapy
Hot water bath is an excellent media to develop hyperthermia
In a 40oC thermal bath, body temperature rises to 39oC in minutes
Physiological stresses ranging from hyperthermia to myocardial ischemia and genetic mutations produce protein damage and misfolded protein structures
Turkish Hungarian Balneotherapy Congress Balçova İzmir 2006

4. How does the cell respond?
Cell respondes to stresses by the rapid synthesis of polypeptides termed (HSPs) or stres proteins
The correct folding of many proteins in a cell requires protein-folding machinery called “molecular chaperones”
HSPs behave as chaperones to repair denatured proteins or promote their degradation after heat shock.
Turkish Hungarian Balneotherapy Congress Balçova İzmir 2006

5. Why heat shock proteins interest us?
Body temperature rise or hyperthermia, causes heat shock protein syntesis in addition to other events
Studies in the last decades suggest that heat shock protein syntesis could occure in physiologically relevant body temperatures
Increased HSP sythesis may be responsible partly from the general effects of balneotherapy??
Turkish Hungarian Balneotherapy Congress Balçova İzmir 2006

6. What Are "Heat Shock" Proteins (HSPs)?
HSPs initially discovered by Ritossa from Italy in Drosphilia Melanogaster larvae that were exposed to “heat shock” in 1962
HSPs are a group of proteins that are induced when a cell undergoes various types of environmental stresses
These proteins are ubiquitous, occurring in all organisms from bacteria and yeast to humans
HSPs present in various forms and are categorized into families on the basis of their molecular weights ranging 10 to 150kDa
HSPs may be present in all cell compartments and are called by molecular sizes as follows: Hsp27, Hsp70, and Hsp90
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HSP Family
Cellular Location
Proposed Function
HSP27 (sHSP)
Cytosol, nucleus
Microfilament stabilization, antiapoptotic
HSP60
Mitochondria
Refolds proteins and prevents aggregation of denatured proteins, proapoptotic
HSP70 family:
Antiapoptotic
  HSP72 (Hsp70)
Protein folding, cytoprotection
  HSP73 (Hsc70)
Molecular chaperones
  HSP75 (mHSP70)
  HSP78 (GRP78) ER
Cytoprotection, molecular chaperones
HSP90
Cytosol, ER, nucleus
Regulation of steroid hormone receptors, protein translocation
HSP110/104
Cytosol
Protein folding
Table  1.   Cellular locations and proposed functions of mammalian heat shock protein familiesHSP, heat shock protein; sHSP, small HSP; ER, endoplasmic reticulum.
Turkish Hungarian Balneotherapy Congress Balçova İzmir 2006

8. What are the functions of HSPs?
HSPs play important physiological roles in both normal cellular homeostasis and systemic or cellular stress such as ischemia, cytokines, and energy depletion
Most HSPs have strong cytoprotective effects,
Behave as molecular chaperones for other cellular proteins
Essential for survival at both normal and elevated temperatures
HSPs also appear to play a critical role in the development of thermotolerance
Turkish Hungarian Balneotherapy Congress Balçova İzmir 2006

9. What are the functions of HSPs?
HSPs also offer the potential to be used as markers of cellular injury and for diagnostic and therapeutic purposes
HSPs may be important modifying factors in an organism's response to a variety of physiologically relevant conditions, such as exercise, hyperthermia, oxidative stress, metabolic challenge, and aging
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A summary of the major pathophysiological signals that activate HSP synthesis (left of vertical solid line) and the potential functions of HSPs (right of vertical solid line)
Benjamin, I. J. et al. Circ Res 1998;83:
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Copyright ©1998 American Heart Association

11. With which ways do HSPs perform these functions?
1) the folding of proteins in various intracellular compartments,
2) the maintenance of structural proteins,
3) the refolding of misfolded proteins,
4) translocation of proteins across membranes and into various cellular compartments,
5) the prevention of protein aggregation, and
6) the degradation of unstable proteins
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The HSP70 family
HSP70 family are the most temperature sensitive and highly conserved of the HSPs
The HSP70s demonstrate a 60-80% base identity among eukaryotic cells
There are at least four distinct proteins in the HSP70 group (HSP72, HSP73, HSP75, and HSP78),
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The HSP70 family
Proteins in the HSP70 group share common protein sequences but are synthesized in response to different stimuli
The 73-kDa protein (HSP73 or Hsc70) is constantly produced (constitutive), whereas the 72-kDa protein (HSP72 or Hsp70) is highly inducible and its synthesis is increased in response to multiple stressors
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Fig. 1. A summary of some of the major physiological signals that activate the inducible form of the 72-kDa heat shock protein (Hsp70) synthesis (top) and a proposed mechanism for increased Hsp70 expression within a cell
Kregel, K. C. J Appl Physiol 92: ;
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Copyright ©2002 American Physiological Society

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Thermotolerance
The first physiological function of the inducible Hsp70 is “acquired thermotolerance”
Which is defined as the ability of a cell or organism to become resistant to heat stress after a prior sublethal heat exposure
Hsp70 is associated with the development of tolerance to a variety of stresses, including hypoxia, ischemia, acidosis, energy depletion, cytokines such as TNF-α and ultraviolet radiation
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Thermotolerance
The phenomenon of acquired thermotolerance is transient in nature
Depends primarily on the severity of the initial heat stress
In general, the greater the initial heat dose, the greater the magnitude and duration of thermotolerance
The expression of thermotolerance following heating will occur within several hours and last 3-5 days in duration
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Thermotolerance
The gut and liver are the first organs to accumulate HSP70 following whole body hyperthermia
HSP synthesis is associated with protection against ischemia-reperfusion injury to the heart, liver, kidney, spinal cord, lungs and light-induced damage to the retina
Studies of cardiac shock followed by resuscitation have revealed that hepatocytes synthesize members of the HSP70 family early in the course of recovery
HSP70 accumulation could be utilized as a biomarker of cellular injury
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Thermotolerance
Proteins in the HSP70 family are involved in preventing protein denaturation and/or processing denatured proteins and protein fragments that are produced by stressors such as hyperthermia
Cells that were made thermotolerant also produce less HSP during a second challenge compared with previously unheated cells
There is a regulation of HSP synthesis that is dependent on the levels of these proteins existing within the cell
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Thermotolerance
The variety of stressors used to condition cells will likely induce other important cellular defense proteins in addition to HSPs, such as antioxidant enzymes
It should also be noted that thermotolerance can be generated in the absence of HSPs
In some studies, thermotolerance was manifested under conditions of a chronic exposure to a lower temperature than is required for HSP accumulation
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20. Functions associated with HSP70
Interestingly, it has also been noted that HSPs can play a role in apoptosis. HSP27, HSP70, and HSP90 proteins are predominantly antiapoptotic, whereas HSP60 is proapoptotic
HSPs serve as modulating signals for immune and inflammatory responses
Elevations in intracellular HSP levels have been shown to improve cell tolerance to inflammatory cytokines such as TNF-α and interleukin-1
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21. Functions associated with HSP70
HSP accumulation may contribute to a reduction in inflammatory cytokine production with cellular challenge
Conversely, when HSPs are present on the surface of cells or released into the local extracellular environment during conditions such as necrotic cell death or viral infection, these proteins have an immune-stimulating response
Hsp70 is also known to facilitate antigen presentation in cells such as macrophages and dendrites
When Hsp70 is applied to the environment external to cells, macrophages and lymphocytes produce inflammatory cytokines
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22. Functions associated with HSP70
Studies have demonstrated the presence of Hsp70 on the surface of tumor cells, potentially functioning as recognition molecules for natural killer (NK) cells
HSPs are important modulators of antigen presentation, T-lymphocyte activation, cytokine production, and NK cell killing
Hsp70 response is markedly reduced with age
Turkish Hungarian Balneotherapy Congress Balçova İzmir 2006

23. Summary and future directions
Whenever a cell — any cell, in any organism — is stressed by heat, cold, ischemia or glucose or oxygen deprivation HSPs are induced
The heat shock response provides thermotolerance to a cell or organism
This response seems not only provide protection from heat, but can, by a mechanism called cross-tolerance, protect against cell death from an entirely different lethal stress (e.g. endotoxin)
Turkish Hungarian Balneotherapy Congress Balçova İzmir 2006

24. Summary and future directions
Mild and nonlethal heat shock (i.e., hyperthermia) is known to protect the myocardium, spinal cord, brain, lungs, pankreas and gut against ischemic injury
Protect retina against photic injury
Protect against infections and inflammatory diseases, aging??
Do hyperthermic balneotherapeutic applications perform thermotolerance???
And may this phenomen partly explain the general effects of balneotherapy???
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References
Calderwood SK, Theriault JR, Gong J: How is the immune response affected by hyperthermia and heat shock proteins? Int J Hyperthermia Dec;21(8):713-6.
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References
Nagarsekar A, Hasday JD, Singh IS: CXC chemokines: A new family of heat-shock proteins? Immunol Invest. 2005;34(3):
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Haveman J, Sminia P, Wondergem J, van der Zee J, Hulshof MC: Effects of hyperthermia on the central nervous system: what was learnt from animal studies? Int J Hyperthermia 2005 Aug;21(5):
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References
Franklin TB, Krueger-Naug AM, Clarke DB, Arrigo AP, Currie RW: The role of heat shock proteins Hsp70 and Hsp27 in cellular protection of the central nervous system. Int J Hyperthermia Aug;21(5):379-92
Tytell M, Hooper PL: Heat shock proteins: new keys to the development of cytoprotective therapies. Expert Opin Ther Targets Apr;5(2):
Kim JH, Kim JH, Yu YS, Jeong SM, Kim KW: Protective effect of heat shock proteins 70.1 and 70.3 on retinal photic injury after systemic hyperthermia. Korean J Ophthalmol Jun;19(2):
Kelly KJ: Heat shock (stress response) proteins and renal ischemia/reperfusion injury. Contrib Nephrol. 2005;148:
Su F, Nguyen ND, Wang Z, Cai Y, Rogiers P, Vincent JL : Fever control in septic shock: beneficial or harmful? Shock Jun;23(6):
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References
Brandhorst D, Olbrich M, Alt A, Bretzel RG, Brandhorst H: Timing of hyperthermic preconditioning affects islet resistance against inflammation. Transplant Proc Jan-Feb;37(1):231-2.
Taylor RP, Benjamin IJ: Small heat shock proteins: a new classification scheme in mammals. J Mol Cell Cardiol Mar;38(3):
Ruell PA, Hoffman KM, Chow CM, Thompson MW: Effect of temperature and duration of hyperthermia on HSP72 induction in rat tissues. Mol Cell Biochem Dec;267(1-2):
Baumeister S, Ofer N, Kleist C, Terne P, Opelz G, Gebhard MM, Germann G, Heitmann C: Reduction of skeletal muscle injury in composite tissue allotransplantation by heat stress preconditioning Plast Reconstr Surg Dec;114(7):
Shinohara T, Takahashi N, Ooie T, Ichinose M, Hara M, Yonemochi H, Saikawa T, Yoshimatsu H: Estrogen inhibits hyperthermia-induced expression of heat-shock protein 72 and cardioprotection against ischemia/reperfusion injury in female rat heart. J Mol Cell Cardiol Nov;37(5):
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References
Kilgore JL: Severity of viral infection is promoted by hyperthermic pretreatment. J Sci Med Sport Jun;7(2):
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Khoei S, Goliaei B, Neshasteh-Riz A, Deizadji A: The role of heat shock protein 70 in the thermoresistance of prostate cancer cell line spheroids. FEBS Lett Mar 12;561(1-3):144-8.
Carini R, Albano E: Recent insights on the mechanisms of liver preconditioning. Gastroenterology Nov;125(5):
Maroni P, Bendinelli P, Tiberio L, Rovetta F, Piccoletti R, Schiaffonati L: In vivo heat-shock response in the brain: signalling pathway and transcription factor activation. Brain Res Mol Brain Res Nov 6;119(1):90-9.
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References
Sasara T, Cizkova D, Mestril R, Galik J, Sugahara K, Marsala M : Spinal heat shock protein (70) expression: effect of spinal ischemia, hyperthermia (42 degrees C)/hypothermia (27 degrees C), NMDA receptor activation and potassium evoked depolarization on the induction. Neurochem Int Jan;44(1):53-64.
Chen HW, Hsu C, Lu TS, Wang SJ, Yang RC: Heat shock pretreatment prevents cardiac mitochondrial dysfunction during sepsis. Shock Sep;20(3):274-9.
Milani V, Noessner E, Ghose S, Kuppner M, Ahrens B, Scharner A, Gastpar R, Issels RD: Heat shock protein 70: role in antigen presentation and immune stimulation. Int J Hyperthermia Nov-Dec;18(6):
Ivor J. Benjamin, , D. Randy McMillan: Stress (Heat Shock) Proteins. Molecular Chaperones in Cardiovascular Biology and Disease. Circulation Research. 1998;83: Kevin C. Kregel: : Molecular Biology of Thermoregulation Invited Review: Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J Appl Physiol 92(5): , 2002;
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References
Larry A. Sonna1,2, Stephen L. Gaffin1, Richard E. Pratt3, Michael L. Cullivan1, Karen C. Angel1, and Craig M. Lilly2 : Molecular Biology of Thermoregulation. Selected Contribution: Effect of acute heat shock on gene expression by human peripheral blood mononuclear cells. J Appl Physiol 92(5): , 2002.
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36. THANK YOU FOR YOUR ATTENTION
Turkish Hungarian Balneotherapy Congress Balçova İzmir 2006