Download Hydrostatic pressure-induced changes in cellular protein synthesis

Biorheology 41 (2004) 309–313
IOS Press
309
Hydrostatic pressure-induced changes
in cellular protein synthesis
Mikko J. Lammi ∗ , Mika A. Elo, Reijo K. Sironen, Hannu M. Karjalainen, Kai Kaarniranta
and Heikki J. Helminen
Department of Anatomy, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland
Abstract. Hydrostatic pressure is a well-known effector of cellular protein synthesis. High continuous hydrostatic pressure
inhibits protein synthesis in general. It has been known for a long time that 30S ribosomal subunit is associated with the effects
of pressure on protein synthesis in prokaryotes, however, the mechanisms of action are still not completely understood. Our
new data suggest that synthesis of eukaryotic elongation factor-2 (eEF-2) is decreased under 30 MPa continuous hydrostatic
pressure. Thus, eEF-2 may have a role in the synthesis of pressure-regulated proteins in eukaryotic cells. The presence of
pressure-sensitive proteins indicate that hydrostatic pressure can induce very specific responses in stressed cells. Accumulation
of heat shock protein 70 and 90 beta occurs under high pressure, independent of the general inhibition of protein synthesis,
although this response appears clearly weaker than during heat stress.
Keywords: Hydrostatic pressure, translation, chondrocyte
1. Introduction
Cells of our body have to sense and withstand various mechanical stresses, such as shearing, compression, tension and osmotic pressure [1]. Hydrostatic pressure is present in various tissues and organs of
our body, the highest levels of the pressure occurring in articular cartilage during locomotion. In chondrocytic cells, numerous cellular events arise in response to cyclic or continuous hydrostatic pressure,
including effects on extracellular matrix production [2–9], modulation of Na+ /H+ exchange [10], transmembrane potentials [11] and cation transport [12], inhibition of shear stress-induced NO release [13]
and cAMP accumulation [14], alterations in actin cytoskeleton [15] and Golgi apparatus [16], and cellular proliferation [17]. This review will summarize our present knowledge of the effects of hydrostatic
pressure on cellular protein synthesis.
2. General effects of hydrostatic pressure on protein synthesis
Protein synthesis is a complicated event that involves a large number of molecules acting together.
Control of mRNA translation is a key regulator of gene expression in eukaryotes [18], and it appears to
be exerted mainly at the stage of initiation. During this event, ribosomal 40S subunit binds to the 5 -end
of mRNA, the start codon is located and ribosomal 60S subunit joins the complex forming a functional
*
Address for correspondence: Mikko Lammi, Department of Anatomy, University of Kuopio, P.O. Box 1627, 70211 Kuopio,
Finland. Tel.: +358 17 163 027; Telefax: +358 17 163 032; E-mail: [email protected].
0006-355X/04/$17.00  2004 – IOS Press and the authors. All rights reserved
310
M.J. Lammi et al. / Hydrostatic pressure-induced changes in cellular protein synthesis
ribosome that enters the elongation stage of translation [19]. In eukaryotes, more than 10 soluble initiation factors participate in the formation of the mRNA recognition and preinitiation complexes [20],
which enable unwinding and scanning of mRNA secondary structure to find a correct initiator AUG
codon [21]. When the translation initiator codon is found, eukaryotic initiation factor-2A (eIF-2A) hydrolyses its bound GTP, and the preinitiation factors will dissociate from the small subunit of ribosome
allowing the elongation of the polypeptide chain to begin.
Phosphorylation events of various eukaryotic initiation factors (eIFs) are involved in the initiation
stage. Phosphorylation of eIF-2A frequently occurs under stressful conditions reducing the translation
rates [22]. Activity of eIF-2B is altered in response to viral infection, hormones, nutrients, growth factors and certain stresses, and it may be regulated directly by its own phosphorylation [23]. eIF-4E also
plays an important role by binding the 5 -cap structure, and it serves to recruit other initiation complex
components to the 5 -end of mRNA [24]. Phosphorylation of eIF-4E is increased by variety of conditions, including exposures of insulin and growth factors [25]. On the other hand, eukaryotic elongation
factor (eEF-2) is inactivated by its phosphorylation during stimuli that, e.g., increase cellular energy
demand or reduce its supply [26]. This protein functions in protein translation facilitating the movement
of peptidyl-tRNA from ribosome A site to the P site [26].
The effects of hydrostatic pressure on translational step of protein synthesis were investigated already
in 1960’s, mainly in bacterial cells [27–29] and later in bacterial cell-free systems [30]. In cell-free
E. coli preparations, a progressive inhibition of protein synthesis was observed starting at approximately
20 MPa that was instantaneously reversible after release of the pressure [31]. Then, the protein synthesis proceeded at a rate similar to that in the non-pressurised control [31]. Studies on whole-cell
preparations of E. coli has shown that 67 MPa hydrostatic pressure totally inhibited protein synthesis
at elongation stage [32]. The response of reticulocyte system was basically similar to prokaryotic systems, although minor differences existed [33]. The response of bacterial protein synthesis to hydrostatic
pressure has been associated with the structure of 30S ribosomal subunit [34,35], while it did not affect
amino acid permeability, aminoacyl-tRNA formation [32], transpeptidation, initiation [31] or ribosome
stability [36].
In our own recent studies, cell cultures of immortalized human chondrocytes [37] and HeLa cells
were labelled for 12 hours with 35 S-methionine and 35 S-cysteine during 30 MPa hydrostatic pressurisation, and the cellular proteins were separated with two-dimensional gel electrophoresis (manuscript
submitted). The pressure inhibited total protein synthesis by approximately 30–40%, suggesting that the
translation machinery was markedly affected by the application of continuous high hydrostatic pressure.
Radiography of two-dimensional gels showed a number of proteins whose relative synthesis level was
altered. The protein spots that changed consistently in both cell types were chosen for mass spectrometric identification. One of the down-regulated proteins could be identified as eEF-2. In Western blot,
a decrease in its amount was observed, while no change in the mRNA level of eEF-2 was evident after
pressurisation. It is known that phosphorylation of eEF-2 prevents its participation in the translocation of
ribosome along mRNA, however, we could not observe increased phosphorylation. It is possible that the
main regulatory mechanism of protein synthesis in pressurised cells acts on inititation factors similarly
to heat stress [38], however, elongation stage may be an additional regulator.
3. Hydrostatic pressure-sensitive proteins
Pressure treatment can result in certain specific responses in the protein expression of chondrocytic
cells. Aggrecan, biglycan, decorin and type II collagen are the most studied molecules in chondro-
M.J. Lammi et al. / Hydrostatic pressure-induced changes in cellular protein synthesis
311
cytic cells. The investigations have mainly shown that intermittent loading at physiological level and
frequencies stimulate the synthesis, while overloading has an inhibitory effect [2–4,9,39,40]. Mechanical compression and hydrostatic loading had similar frequency-dependent stimulation of proteoglycan synthesis in tissue explants, while monolayer cultures required longer loading period to be stimulated [39,41].
High continuous hydrostatic pressure increases especially the accumulation of heat shock protein 70
in the pressurised cells [42–45]. The synthesis of heat shock protein 90 beta was also induced in immortalised human chondrocytes and HeLa cells, while the alpha form remained at the control level [44,
46]. It is noteworthy that in bovine primary chondrocytes no heat shock protein response was detected,
while, e.g., primary fibroblast and synovial cells responded strongly under pressure [47]. Although the
mechanism behind this barotolerance is not known [48], the result may be interpreted to indicate that the
loading history of the cells may affect their barotolerance. Interestingly, barotolerance of bacteria differs
in various strains, and appears to be associated with the structure of 30S ribosomal subunit [34].
The effects of hydrostatic pressure on various proteins have been mainly investigated at mRNA level.
High pressure induces the expression of interleukin-6, tumor necrosis factor-alpha and decreases transforming growth factor-beta mRNA levels [42,49]. In intervertebral disc, a physiological level of hydrostatic pressure had an anabolic influence on the tissue, while pressure levels of 3 MPa or higher caused
a catabolic effect [50]. Recently, a cDNA array data showed that intermittent and static 5 MPa pressure
had differential effects in chondrosarcoma cells [51]. High hydrostatic pressure revealed other specific
cDNA array changes, of which the decreased mRNA and protein level of Id-1 and -3 proteins are interesting [52], since they are capable of directing cellular differentiation.
4. Messenger RNA stabilisation by high continuous hydrostatic pressure
High continuous pressure promotes an interesting cell biological phenomenon by increasing hsp70
mRNA stability, without any detectable induction of the actual gene activity [43]. High pressure may
have selective effect on mRNA stability. It has been previously reported that histone mRNA levels were
reduced partly because of suppression of transcription (one third) and partly due to loss of mRNA stability (two thirds) [53]. A recent cDNA study confirmed that sensitivity of mRNA stabilisation varies
among the different transcripts [54]. Thus, high hydrostatic pressure may turn out an useful tool when
the mechanisms of mRNA stabilisation are investigated.
References
[1] M.J. Lammi, R.K. Sironen, M.A. Elo, N. Oksala, K. Kaarniranta, H.M. Karjalainen and H.J. Helminen, Responses of
mammalian cells to mechanical forces, in: Recent Research Developments in Biophysics and Biochemistry, Vol. 1. Research Signpost, Trivandrum, India, 2001, pp. 77–98.
[2] G.P. van Kampen, J.P. Veldhuijzen, R. Kuijer, R.J. van de Stadt and C.A. Schipper, Cartilage response to mechanical force
in high-density chondrocyte cultures, Arthritis Rheum. 28 (1985), 419–424.
[3] L. Lippiello, C. Kaye, T. Neumata and H.J. Mankin, In vitro metabolic response of articular cartilage segments to low
levels of hydrostatic pressure, Connect. Tiss. Res. 13 (1985), 99–107.
[4] A.C. Hall, J.P. Urban and K.A. Gehl, The effects of hydrostatic pressure on matrix synthesis in articular cartilage, J. Orthop. Res. 9 (1991), 1–10.
[5] F. Lafeber, J.P. Veldhuijzen, J.L. Vanroy, O. Huber-Bruning and J.W. Bijlsma, Intermittent hydrostatic compressive force
stimulates exclusively the proteoglycan synthesis of osteoarthritic human cartilage, Br. J. Rheumatol. 31 (1992), 437–442.
312
M.J. Lammi et al. / Hydrostatic pressure-induced changes in cellular protein synthesis
[6] M.J. Lammi, R. Inkinen, J.J. Parkkinen, T. Häkkinen, M. Jortikka, L.O. Nelimarkka, H.T. Järveläinen and M.I. Tammi,
Expression of reduced amounts of structurally altered aggrecan in articular cartilage chondrocytes exposed to high hydrostatic pressure, Biochem. J. 304 (1994), 723–730.
[7] R.L. Smith, S.F. Rusk, B.E. Ellison, P. Wessells, K. Tsuchiya, D.R. Carter, W.E. Caler, L.J. Sandell and D.J. Schurman,
In vitro stimulation of articular chondrocyte mRNA and extracellular matrix synthesis by hydrostatic pressure, J. Orthop.
Res. 14 (1996), 53–60.
[8] M.O. Jortikka, J.J. Parkkinen, R.I. Inkinen, J. Kärner, H.T. Järveläinen, L.O. Nelimarkka, M.I. Tammi and M.J. Lammi,
The role of microtubules in the regulation of proteoglycan synthesis in chondrocytes under hydrostatic pressure, Arch.
Biochem. Biophys. 374 (2000), 172–180.
[9] T. Ikenoue, M.C. Trindade, M.S. Lee, E.Y. Lin, D.J. Schurman, S.B. Goodman and R.L. Smith, Mechanoregulation of human articular chondrocyte aggrecan and type II collagen expression by intermittent hydrostatic pressure in vitro, J. Orthop.
Res. 21 (2003), 110–116.
[10] J.A. Browning, R.E. Walker, A.C. Hall and R.J. Wilkins, Modulation of Na+ x H+ exchange by hydrostatic pressure in
isolated bovine articular chondrocytes, Acta Physiol. Scand. 166 (1999), 39–45.
[11] M.O. Wright, R.A. Stockwell and G. Nuki, Response of plasma membrane to applied hydrostatic pressure in chondrocytes
and fibroblasts, Connect. Tiss. Res. 28 (1992), 49–70.
[12] A.C. Hall, Differential effects of hydrostatic pressure on cation transport pathways of isolated articular chondrocytes,
J. Cell. Physiol. 178 (1999), 197–204.
[13] M.S. Lee, M.C. Trindade, T. Ikenoue, D.J. Schurman, S.B. Goodman and R.L. Smith, Intermittent hydrostatic pressure
inhibits shear stress-induced nitric oxide release in human osteoarthritic chondrocytes in vitro, J. Rheumatol. 30 (2003),
326–328.
[14] L.A. Bourret and G.A. Rodan, Inhibition of cAMP accumulation in epiphyseal cartilage cells exposed to physiological
pressure, Calcif. Tiss. Res. 21(Suppl.) (1976), 431–436.
[15] J.J. Parkkinen, M.J. Lammi, R. Inkinen, M. Jortikka, M. Tammi, I. Virtanen and H.J. Helminen, Influence of short-term
hydrostatic pressure on organization of stress fibers in cultured chondrocytes, J. Orthop. Res. 13 (1995), 495–502.
[16] J.J. Parkkinen, M.J. Lammi, A. Pelttari, H.J. Helminen, M. Tammi and I. Virtanen, Altered Golgi apparatus in hydrostatically loaded articular cartilage chondrocytes, Ann. Rheum. Dis. 52 (1993), 192–198.
[17] D.B. Saris, A. Sanyal, K.N. An, J.S. Fitzsimmons and S.W. O’Driscoll, Periosteum responds to dynamic fluid pressure by
proliferating in vitro, J. Orthop. Res. 17 (1999), 668–677.
[18] T.E. Dever, Gene-specific regulation by general translation factors, Cell 108 (2002), 545–556.
[19] G.S. Wilkie, K.S. Dickson and N.K. Gray, Regulation of mRNA translation by 5 - and 3 -UTR-binding factors, Trends
Biochem. Sci. 28 (2003), 182–188.
[20] T.V. Pestova, V.G. Kolupaeva, I.B. Lomakin, E.V. Pilipenko, I.N. Shatsky, V.I. Agol and C.U. Hellen, Molecular mechanisms of translation initiation in eukaryotes, Proc. Natl. Acad. Sci. USA 98 (2001), 7029–7036.
[21] M. Kozak, Pushing the limits of the scanning mechanism for initiation of translation, Gene 299 (2002), 1–34.
[22] M.J. Clemens, Initiation factor eIF2 alpha phosphorylation in stress responses and apoptosis, Prog. Mol. Subcell. Biol. 27
(2001), 57–89.
[23] B.L. Webb and C.G. Proud, Eukaryotic initiation factor 2B (eIF2B), Int. J. Biochem. Cell Biol. 29 (1997), 1127–1131.
[24] G.C. Scheper and C.G. Proud, Does phosphorylation of the cap-binding protein eIF4E play a role in translation initiation?,
Eur. J. Biochem. 269 (2002), 5350–5359.
[25] A. Flynn and C.G. Proud, Insulin and phorbol ester stimulate initiation factor eIF-4E phosphorylation by distinct pathways
in Chinese hamster ovary cells overexpressing the insulin receptor, Eur. J. Biochem. 236 (1996), 40–47.
[26] G.J. Browne and C.G. Proud, Regulation of peptide-chain elongation in mammalian cells, Eur. J. Biochem. 269 (2002),
5360–5368.
[27] E.C. Pollard and P.K. Weller, Effect of hydrostatic pressure on the synthetic processes in bacteria, Bibl. Laeger 112 (1966),
573–580.
[28] J.V. Landau, Protein and nucleic acid synthesis in Escherichia coli: pressure and temperature effects, Science 153 (1966),
1273–1274.
[29] J.V. Landau, Induction, transcription and translation in Escherichia coli: a hydrostatic pressure study, Biochim. Biophys.
Acta 149 (1967), 506–512.
[30] R.M. Arnold and L.J. Albright, Hydrostatic pressure effects on the translation stages of protein synthesis in a cell-free
system from Escherichia coli, Biochim. Biophys. Acta 238 (1971), 347–354.
[31] J.R. Schwarz and J.V. Landau, Inhibition of cell-free protein synthesis by hydrostatic pressure, J. Bacteriol. 112 (1972),
1222–1227.
[32] J.R. Schwarz and J.V. Landau, Hydrostatic pressure effects on Escherichia coli: site of inhibition of protein synthesis,
J. Bacteriol. 109 (1972), 945–948.
[33] A.C. Scheck and J.V. Landau, The stability of rabbit reticulocyte polysomes at high hydrostatic pressure, Biochim. Biophys. Acta 699 (1982), 226–231.
M.J. Lammi et al. / Hydrostatic pressure-induced changes in cellular protein synthesis
313
[34] W. Smith, D. Pope and J.V. Landau, Role of bacterial ribosome subunits in barotolerance, J. Bacteriol. 124 (1975), 582–
584.
[35] J.V. Landau, W.P. Smith and D.H. Pope, Role of the 30S ribosomal subunit, initiation factors, and specific ion concentration in barotolerant protein synthesis in Pseudomonas bathycetes, J. Bacteriol. 130 (1977), 154–159.
[36] D.H. Pope, N.T. Connors and J.V. Landau, Stability of Escherichia coli polysomes at high hydrostatic pressure, J. Bacteriol. 121 (1975), 753–758.
[37] M.B. Goldring, J.R. Birkhead, L.F. Suen, R. Yamin, S. Mizuno, J. Glowacki, J.L. Arbiser and J.F. Apperley, Interleukin-1
beta-modulated gene expression in immortalized human chondrocytes, J. Clin. Invest. 94 (1994), 2307–2316.
[38] R. Panniers, Translational control during heat shock, Biochimie 76 (1994), 737–747.
[39] J.J. Parkkinen, J. Ikonen, M.J. Lammi, J. Laakkonen, M. Tammi and H.J. Helminen, Effects of cyclic hydrostatic pressure
on proteoglycan synthesis in cultured chondrocytes and articular cartilage explants, Arch. Biochem. Biophys. 300 (1993),
458–465.
[40] R.L. Smith, J. Lin, M.C. Trindade, J. Shida, G. Kajiyama, T. Vu, A.R. Hoffman, M.C. van der Meulen, S.B. Goodman,
D.J. Schurman and D.R. Carter, Time-dependent effects of intermittent hydrostatic pressure on articular chondrocyte type
II collagen and aggrecan mRNA expression, J. Rehabil. Res. Dev. 37 (2000), 153–161.
[41] J.J. Parkkinen, M.J. Lammi, H.J. Helminen and M. Tammi, Local stimulation of proteoglycan synthesis in articular cartilage explants by dynamic compression in vitro, J. Orthop. Res. 10 (1992), 610–620.
[42] K. Takahashi, T. Kubo, K. Kobayashi, J. Imanishi, M. Takigawa, Y. Arai and Y. Hirasawa, Hydrostatic pressure influences
mRNA expression of transforming growth factor-beta 1 and heat shock protein 70 in chondrocyte-like cell line, J. Orthop.
Res. 15 (1997), 150–158.
[43] K. Kaarniranta, M. Elo, R. Sironen, M.J. Lammi, M.B. Goldring, J.E. Eriksson, L. Sistonen and H.J. Helminen, Hsp70
accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization
rather than transcriptional activation, Proc. Natl. Acad. Sci. USA 95 (1998), 2319–2324.
[44] M.A. Elo, R.K. Sironen, K. Kaarniranta, S. Auriola, H.J. Helminen and M.J. Lammi, Differential regulation of stress
proteins by high hydrostatic pressure, heat shock, and unbalanced calcium homeostasis in chondrocytic cells, J. Cell.
Biochem. 79 (2000), 610–619.
[45] R. Sironen, M. Elo, K. Kaarniranta, H.J. Helminen and M.J. Lammi, Transcriptional activation in chondrocytes submitted
to hydrostatic pressure, Biorheology 37 (2000), 85–93.
[46] M.A. Elo, R.K. Sironen, H.M. Karjalainen, K. Kaarniranta, H.J. Helminen and M.J. Lammi, Specific induction of heat
shock protein 90beta by high hydrostatic pressure, Biorheology 40 (2003), 141–146.
[47] K. Kaarniranta, C.I. Holmberg, M.J. Lammi, J.E. Eriksson, L. Sistonen and H.J. Helminen, Primary chondrocytes resist hydrostatic pressure-induced stress while primary synovial cells and fibroblasts show modified Hsp70 response, Osteoarthritis Cartilage 9 (2001), 7–13.
[48] K. Kaarniranta, C.I. Holmberg, H.J. Helminen, J.E. Eriksson, L. Sistonen and M.J. Lammi, Protein synthesis is required
for stabilization of hsp70 mRNA upon exposure to both hydrostatic pressurization and elevated temperature, FEBS Lett.
475 (2000), 283–286.
[49] K. Takahashi, T. Kubo, Y. Arai, I. Kitajima, M. Takigawa, J. Imanishi and Y. Hirasawa, Hydrostatic pressure induces
expression of interleukin 6 and tumour necrosis factor alpha mRNAs in a chondrocyte-like cell line, Ann. Rheum. Dis. 57
(1998), 231–236.
[50] T. Handa, H. Ishihara, H. Ohshima, R. Osada, H. Tsuji and K. Obata, Effects of hydrostatic pressure on matrix synthesis
and matrix metalloproteinase production in the human lumbar intervertebral disc, Spine 22 (1997), 1085–1091.
[51] H.M. Karjalainen, R.K. Sironen, M.A. Elo, K. Kaarniranta, M. Takigawa, H.J. Helminen and M.J. Lammi, Gene expression profiles in chondrosarcoma cells subjected to cyclic stretching and hydrostatic pressure. A cDNA array study,
Biorheology 40 (2003), 93–100.
[52] R.K. Sironen, H.M. Karjalainen, M.A. Elo, K. Kaarniranta, K. Törrönen, M. Takigawa, H.J. Helminen and M.J. Lammi,
cDNA array reveals mechanosensitive genes in chondrocytic cells under hydrostatic pressure, Biochim. Biophys. Acta
1591 (2002), 45–54.
[53] A.L. Symington, S. Zimmerman, J. Stein, G. Stein and A.M. Zimmerman, Hydrostatic pressure influences histone mRNA,
J. Cell Sci. 98 (1991), 123–129.
[54] R.K. Sironen, H.M. Karjalainen, K. Törrönen, M.A. Elo, K. Kaarniranta, M. Takigawa, H.J. Helminen and M.J. Lammi,
High pressure effects on cellular mRNA stability. A cDNA array analysis, Biorheology 39 (2002), 111–117.