Trehalose improves cell proliferaTion and dehydraTion Tolerance of human hacaT cells

Trehalose is a disaccharide molecule that serves as a natural osmotic regulator in halophilic microorganisms and plants but not in mammals. We observed that human HaCaT cells supplied with trehalose improved cell proliferation and extended viability under dehydration. In HaCaT cells, in response to increasing concentrations of exogenous trehalose, the levels of heat shock protein (HSP) 70 increased and matrix metalloproteinase (MMP) 1 decreased. Proteome analysis of trehalose-treated HaCaT cells revealed remarkable increases in the levels of proteins involved in cell signaling and the cell cycle, including p21 activated kinase I, Sec I family domain protein and elongation factor G. Moreover, the proteins for cell stress resistance, tryptophan hydroxylase, serine/cysteine proteinase inhibitors and vitamin D receptors were also increased. In addition, the proteins responsible for the maintenance of the cytoskeleton and cellular structures including procollagen-lysine dioxygenase, vinculin and ezrin were increased. Proteomic data revealed that trehalose affected HaCaT cells by inducing the proteins involved in cell proliferation. These results suggest that trehalose improves the proliferation and dehydration tolerance of HaCaT cells by inducing proteins involved in cell growth and dehydration protection.

Owing to its ability to prevent starch degradation and stabilize proteins and lipids, trehalose is widely used by a variety of industries (Crowe, 2007).For bio-industrial purposes, trehalose has also been investigated for its ability to inhibit osteoclast differentiation (Arai et al., 2001), stabilize drug molecules (Jain and Roy, 2008) and preserve vaccines and liposomes (Crowe, 2007), as well as for its potential for cryoprotection of human fibroblasts and oocytes (Guo et al., 2000;Ma et al., 2005).Recent studies have reported that trehalose protects against hypoxia in mammalian cells and anoxia-induced protein aggregation (Chen and Haddad, 2004).Furthermore, trehalose inhibits the aggregation and neurotoxicity of beta amyloid protein in Alzheimer's disease (Liu et al., 2005).In prion research, trehalose weakened the aggregation of PrP Sc molecules, resulting in the protection of prion-infected cells against oxidative inTroducTion Trehalose (α-d-glucopyranosyl-α-d-glucopyranoside) is a non-reducing disaccharide with two units of glucose molecules that are linked together in a α-1,1glycosidic linkage.Trehalose is present in diverse organisms, including bacteria, fungi, invertebrates and plants (Elbein et al., 2003).The physiological functions of trehalose include osmotic regulation to provide protection against drought and to provide salt and cold tolerance to microorganisms and plants (Kempf and Bremer, 1998;Reina-Bueno et al., 2012).Trehalose has also been reported to act as a compatible solute in halophilic bacteria, cyanobacteria and yeasts (Arguelles, 2000), as well as being a carbon source (Thevelein, 1984), desiccation protectant (Crowe, 2002), immunogenicity agent (Rao et al., 2006) and plant growth regulator (Rolland et al., 2002).damage (Beranger et al., 2008).Additionally, studies of trehalose in biotechnology have demonstrated its importance in plant and microbial stress responses (Hincha and Hagemann, 2004).In applications for plant biotechnology, transgenic plants harboring microbial trehalose biosynthesis genes showed strong resistance against drought, salt and cold tolerance in response to increased levels of trehalose (Garg et al., 2002;Lee et al., 2012).Overall, trehalose is currently being utilized in biotechnology, but its cellular effects in human cells are not well understood.Therefore, in this study, we investigated the proteomic and cellular dynamics of trehalose in the human HaCaT cells.

cell proliferation assay
Cell viability and proliferation were measured by the MTT assay after treatment with appropriate concentrations of trehalose for the specified time periods.Briefly, cells were harvested with trypsin EDTA and then washed twice with PBS, after which they were resuspended in 1 mL of fresh DMEM medium.Germany).Additionally, 20 μl aliquots of the cells were counted using a hemocytometer under a light microscope (IX 70, Olympus, Japan).

dehydration tolerance assay
Approximately 5×10 4 HaCaT cells were seeded on 6-well plates in DMEM medium with 1% (v/v) trehalose and then grown for 120 h.The medium was then removed from the HaCaT cell cultured plates with PBS two times.Dehydration tolerance was assayed by removing media as previously described (Guo et al., 2000).The PBS was then completely removed, after which the plates were sealed in plastic wrap and stored in a growth chamber at 37°C, 5% CO 2 .At 15, 30 and 60 min intervals, the cells were rescued by adding DMEM medium.After 5 min of stabilization, the cell viability was assayed using a Cell Proliferation Kit (Roche Applied Science).

Western blot analysis
HaCaT cells treated with DMEM medium containing 0.01, 0.1 and 1% trehalose for 120 h were harvested with trypsin and then washed once with 10 mM PBS (pH 7.4) containing 150 mM NaCl, after which they were lysed in PBS containing 0.1% SDS and 10 mM β mercaptoethanol.Next, the lysates containing proteins were applied to 10% SDS-polyacrylamide gels for electrophoresis.After electrophoresis, they were transferred to nitrocellulose membranes in 20% methanol/25 mM Tris/192 mM glycine.Membranes were then blocked with 5% non-fat dry milk in TTBS (25 mM Tris-HCl, 150 mM NaCl and 0.2% Tween-20) and probed by incubation with rabbit polyclonal antibodies against human interleukin-1α (IL-1α), HSP70, MMP1 and actin (1:15000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h.The membranes were subsequently rinsed three times in TTBS, after which they were incubated for 1 h with an anti-rabbit HRP-antibody as a secondary antibody (Santa Cruz Biotechnology) at 1:10000 for 1 h.The membranes were then rinsed in TTBS, treated with the Western Lightning Chemiluminescence reagent ECL system (Amersham Biosciences Inc., NJ, USA), and visualized using X-ray film.

resulTs exogenous trehalose is nontoxic to hacaT cells
To test the toxicity of trehalose to human cells, Ha-CaT cells were seeded at a density of 3×10 4 cells per well in a 6-well plate treated with 0.01%, 0.1% and 1% exogenous trehalose and grown for the designated periods (Fig. 1A).After seeding, fresh medium was supplied every 48 h.Colonies of treated and non-treated HaCaT cells showed a single layer, then expanded to a round shape and eventually covered the culture plate.Cells grew in a single layer on each plate for 120 h and underwent senescence within 264 h (Fig. 1A).There were no differences observed in cell phenotype or growth patterns between trehalose-treated or nontreated plates during the experimental time periods.
Overall, phenotypic analysis revealed no notable cell death in response to trehalose.

Trehalose increased the proliferation of hacaT cells
After adding appropriate amounts of trehalose to the culture media, cells were counted to evaluate cell proliferation (Fig. 1B).There were no differences in the numbers of cells between groups at 72 h.However, at 120 h after treatment, the number of cells in the trehalose-treated group was higher than in the nontreated group, with an average of 6.0×10 6 cells being present in the 0.01% trehalose group, but only 4.8×10 6 cells in the non-treated group (Fig. 1B).Additionally, about 5.0×10 6 cells were present in the 0.1% and 1% trehalose-containing medium.Overall, the cell proliferation experiments indicated that the numbers of HaCaT cells increased by almost 25% in response to treatment with 0.01% trehalose, and by more than 4% in response to treatment with 0.1% and 1% trehalose, relative to control cells without trehalose.

Trehalose affected the expression of hsp70, il-1α and mmp1
Exogenous treatment of cells with small molecules is known to generally result in stress; therefore, we examined whether exogenous trehalose caused stress in HaCaT cells by measuring the changes in stressrelated proteins, including HSP70, IL-1α and MMP1 (Fig. 2).Levels of HSP70 proteins increased in Ha-CaT cells treated with 0.01%, 0.1% and 1% trehalose compared to the control (Fig. 2).HSP70 transcripts were also slightly upregulated in response to increasing concentrations of trehalose (Fig. 3).However, the amount of IL-1α and MMP1 proteins decreased slightly in response to increasing trehalose concentrations (Fig. 2).Additionally, RT-PCR revealed that the transcripts of IL-1α and MMP1 decreased (Fig. 3).

Trehalose-containing medium improved dehydration tolerance of hacaT cells
To determine if HaCaT cells in medium containing trehalose showed resistance to drying and cell death, cells were seeded in 6-well plates at a density of 3×10 5 cells/well (Fig. 4) and then cultivated for 120 h with or without 1% trehalose.After observing confluent growth with a single layer on culture plates, culture medium was removed from each well of the plates, after which they were then sealed in plastic wrap and incubated for 15, 30 or 60 min in a growth chamber at 37°C, 5% CO 2 .To measure cell viability after removing the medium, fresh culture medium without trehalose was added after incubation in the dry state for the specified period.The viability of the rehydrated cells was then determined by MTT assay.Trehalose-treated and untreated cells showed complete viability in each experimental group before removing the medium (Fig. 4).At 15 min after removal of the medium, the cell viability in the 1% trehalose medium remained at 90%, but that of the non-trehalosetreated cells was only 30%.At 30 min after removing the medium, about 20% of the HaCaT cells were still viable in the medium with 1% trehalose, but most of the cells grown in medium without trehalose were non-viable.At 60 min after removing the medium, no cells were viable in either group (Fig. 4).

proteome profiling of hacaT cells
We conducted a proteomic study to investigate whether the patterns of protein expression differed between control cells and 0.01% trehalose-treated cells.Specifically, two 2-D gels were generated from the proteins of trehalose-treated or untreated HaCaT cells (Fig. 5).

fig. 4.
Cell viability assay of treatment with 1% trehalose followed by culture under different drying conditions.(A) Pictures of cell growth after removal of culture media for the indicated times.(B) Viable numbers of cells treated with and without 1% trehalose were determined after the culture media had been removed for the indicated times.Cell viability was measured by MTT assay.
The differentially expressed protein spots between two experimental groups were normalized based on total spot intensity.The relative intensities of the spots differentially expressed between treated and untreated cells are shown in Fig. 3, and differentially expressed spots were identified and listed in Table 1.Approximately 800 protein spots were reproducibly detected in gels in both biological replicates, approximately 159 of which were differentially expressed when trehalose treated and nontreated cells were compared.Among these, 114 protein spots were upregulated and 45 were downregulated.Additionally, 40 protein spots in trehalose-treated cells were newly induced relative to the non-treated cells (Fig. 5).These identified protein spots represent known proteins as well as five unknown proteins (Table 1 and Fig. 5).The identified proteins included some associated with cell morphology and proliferation.Tryptophan hydroxylase (spot ID-7103R) was upregulated in trehalose-treated cells, as was vitamin D receptor (spot ID-7701R), p21-activated kinase 2, a tumor repression protein, (spot ID-6501R) and vinculin isoform (spot ID-6704R) (Table 1).Additionally, proteins associated with cytoskeletons, including ezrin (spot ID-7604R), procollagen-lysine 2-oxoglutarate 5-dioxygenase 3 (spot ID-5612R) and integrin alpha 6 (spot ID-4709R), were upregulated in trehalose-treated cells relative to nontreated cells.Two proteins, tumor endothelial marker 2 (spot ID-7012R) and an unknown protein (spot ID-5007R), were not identified in cells treated with trehalose (Table 1 and Fig. 5).

Trehalose stabilizes cellular proteins for dehydration tolerance
In this study, we found that trehalose extended dehydration tolerance and increased the cell proliferation rate of HaCaT cells.Additionally, we observed that the level of HSP70 proteins increased gradually as the trehalose concentration in the medium increased from 0.01% to 1%.These findings indicate that trehalose may induce stress response proteins such as molecular chaperone components.Similar findings in a previous study demonstrated that dur-ing heat shock in yeast, trehalose was induced and stabilized the partially folded proteins, resulting in rapid hydrolyzation of the active form of proteins by molecular chaperones (Crowe, 2007).In nature, trehalose is known to be an important small molecule that maintains osmotic balance to protect bacteria and plants from environmental desiccation (Kempf and Bremer 1998;Crowe LM 2002;Garg et al., 2002;Lee et al., 2012).Interestingly, induction of trehalose biosynthesis in human primary fibroblasts by the expression of otsA and otsB genes of E. coli was found to increase cell viability in the dry state (Guo et al., 2000), which is in line with the results of the present study.Another report previously demonstrated that trehalose maintained the vitality of mouse epithelial cells in vitro (Qu et al., 2014).These findings indicate that trehalose may bind water molecules, thereby protecting cytosolic proteins, providing protection against dehydration.Overall, the results of this study revealed that trehalose improved dehydration tolerance of human HaCaT cells.

Trehalose may provide protective effects to hacaT cells
Interleukin-1α is present in normal keratinocytes of the skin and the epithelial cells of healthy individuals.Moreover, IL1-α is a known molecular marker of inflammation processes.As shown in Fig. 2, the levels of IL-1α protein did not change, but decreased slightly in response to the addition of up to 1% trehalose to the medium.These findings suggest that exogenous trehalose is not an allergen for allergenic response, and is similar to the previously reported suppression of hemolysate-induced production of TNF-α, IL-6 and IL-1α in response to trehalose (Echigo et al., 2012).The level of MMP1 was reduced by almost 20% in cells grown in 1% trehalose-containing medium relative to those grown without trehalose treatment.Similarly, transcripts of MMP1 were reduced in response to increasing concentrations of trehalose.MMP1 is known to degrade types I, II and III collagen, resulting in age-related wrinkling of skin (Jones et al., 2003;Kim et al., 2005).These findings suggest that trehalose can be applied widely in health and cosmetic industry including anti-aging preparations.

proteins responded by trehalose
The proteome provided useful information concerning how trehalose affects cell proliferation.Several proteins revealing notable changes in expression in response to trehalose were selected and their functions are discussed below.Protein spot 7103R was identified as tryptophan hydroxylase (TPH).This protein was upregulated by as much as 300-fold in trehalose-treated cells, but was not identified in the control cells.TPH is the rate-limiting enzyme for the synthesis of serotonin in vivo (Mockus and Vrana 1998).Serotonin is an important molecule for neural transmission, an antioxidant and a mitogen.Therefore, TPH induction by trehalose likely induced serotonin synthesis in cells, resulting in reduced oxidative stresses and activation of cell proliferation.
Protein spot 7701R was identified as the vitamin D receptor (VDR).The VDR protein was upregulated 7-fold and highly induced in the trehalose-treated cells compared to the control.The VDR is a major receptor for vitamin D that activates the vitamin D endocrine system (Bouillon et al., 2008), and vitamin D functions as a hormone to promote differentiation of epidermal keratinocytes.Therefore, trehalose induced the VDR, resulting in promotion of the proliferation of HaCaT cells.
Protein spot 6501R was identified as p21 activated kinase (PAK), which is a critical effector that links Rho GTPases to cytoskeleton reorganization and nuclear signaling (Knaus et al., 1995).The PAK in trehalosetreated keratinocytes was upregulated 5-fold relative to the control.PAK serves as an important regulator of cytoskeletal dynamics and cell motility; therefore, we propose that trehalose-induced PAK proteins resulted in the progression of cell proliferation and maintenance of cell persistence under dehydration.
Protein spot 6704R was identified as human vinculin protein (VCL), a cytoskeletal protein that localizes at the cytoplasmic face of the cell matrix, where it enables cell-cell adhesions and is involved in anchoring F-actin to the membrane, resulting in strong cell adhesion with structural plasticity (Palmer et al., 2009).The adherence among cells and the extracellular matrix is essential to the maintenance of tissue integrity.Proteomic results revealed that vinculin was highly upregulated by trehalose, and that it could promote cells to adhere tightly to one another and prevent dehydration stress.
Protein spot 6616R is a member of the Sec1 family domain containing I (SCFD1), known as Sly1 protein (Bando et al., 2005).Many studies have suggested that Sly1 plays an important cytoprotective role in dopaminergic neuronal cell death (Bando et al., 2005).Thus, Sly1 induction in trehalose-treated cells could contribute to tolerance against dehydration stress.
Protein spot 7607R is a P1cdc47 protein (Table 1), which is also known as CDC47 (Sakwe et al., 2007).The major function of CDC47 is participation in the regulation of mammalian DNA replication.Human CDC47 was recently shown to function as a component of the regulatory mechanism in cell proliferation; therefore, hCDC47 protein levels in cells have been used as a cell proliferation index (Sakwe et al., 2007).Proteomic analysis revealed increasing levels of hCDC47 protein in HaCaT cells treated with tre-halose, providing evidence that trehalose led to an increased proliferation rate of these cells.
Protein spot 2702 was identified as a subunit of the 2-oxoglutarate dehydrogenase (OGDH), which is another name for the alpha-ketoglutarate dehydrogenase complex (KGDHC) (Szabo et al., 1994).Since KGDHC is a key and rate-limiting enzyme of the TCA cycle, the abundance of this complex in HaCaT cells grown in trehalose might mediate the dynamics of cellular processes required for active cell proliferation.
Protein spot ID 7604R was identified as the ezrin protein.As a member of the ezrin/radixin/moesin (ERM) protein family, the ezrin protein serves as an intermediate between the plasma membrane and the actin cytoskeleton (Louvet-Vallée, 2000).Therefore, we assumed that ezrin protein induced by trehalose increases and serves to form a more rigid cell membrane surface, as well as enabling cell surface adhesion among cells to maintain osmotic balance under dehydration.
Protein spot 5612R is procollagen-lysine,2-oxoglutarate 5-dioxygenase 3 (EC 1.14.11.4), which is known as procollagen-lysine 5-dioxygenase or lysyl hydroxylase (LH).The enzyme with cofactors iron and ascorbate catalyzes the hydroxylation of lysyl residues in collagen peptides, which are associated with collagen biosynthesis.The function of this dioxygenase is essential to the stability of the intermolecular collagen crosslinks to maintain cell growth and viability (Wang et al., 2009).Therefore, we suggest that trehalose induction of this dioxygenase stabilized collagen peptides, resulting in resistance to dehydration stress.
Protein spot 4709R is integrin alpha 6 (ITGA6).Integrins are integral cell-surface proteins composed of an alpha chain and a beta chain that participate in cell adhesion as well as cell-surface mediated signaling (Livshitset al., 2012).The abundance of ITGA6 in trehalose-treated cells may result in increased proliferation and cell structural rigidity to dehydration.
Several proteins were downregulated in HaCaT cells treated with trehalose: SUMF2 protein (5112R), proteasome alpha 6 subunit (7006R), chorionic somatomammotropin hormone 1 (5005R) and tumor endothelial marker 2 (7012R).Although the function of these proteins is not clearly understood, the changes in their abundance in trehalose-treated cells may mediate cellular dynamics.
The results presented here suggest that trehalose improves the proliferation and dehydration tolerance of HaCaT cells by inducing many of the proteins involved in cell growth and dehydration protection.Our results are compatible with the use of trehalose in cryoprotection of animal cells and tissues, preservation of proteins, including antibodies and engineered enzymes, and its various applications in medicine, cosmetics and biotechnology (Guo et al., 2000;Arai et al., 2001;Chen and Haddad, 2004;Ma et al., 2005;Crowe, 2007;Echigo et al., 2012).
fig. 1. Cell proliferation assay following treatment with trehalose.(A) Pictures of cultures containing 0, 0.01, 0.1 and 1% trehalose in the medium on the given days.HaCaT cells were seeded before trehalose was added.(B) Cells were enumerated after the indicated time periods.

fig. 2 .
fig. 2. Changes in IL-1α, HSP70 and MMP1 protein levels.(A) Western blot analysis of the changes in IL-1α, HSP70, MMP1 and actin levels.(B) Arbitrary numbers obtained upon densitometric analysis of IL-1α, HSP70, MMP1 and actin proteins evaluated by Western blot analysis in panel (A) were measured.AU, arbitrary units.

fig. 3 .
fig. 3. Changes in levels of IL-1α, HSP70 and MMP1 transcripts.(A) RT-PCR analysis of the changes in IL-1α, HSP70, MMP1 and actin transcripts.(B) Arbitrary signals of IL-1α, HSP70, MMP1 and actin gene PCR products evaluated in panel (A) were measured.AS, arbitrary unit of signals.

fig. 5 .
fig. 5. Differentially expressed protein spots induced by trehalose.(A) Protein spots with the spot number on the gels showing higher or lower levels of expression in non-treated control (C) and 0.01% trehalose-treated (TH) cells.(B) The selected proteins of differentially expressed spots in the 2-D protein gels between control (white bars) and trehalose (gray bars) treated HaCaT cells.Values are the means ± SD of proteins from two independent experiments.The highest value was set at 3,000.The white bars indicate non-treated control cells and the gray bars indicate trehalose treated cells.