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Cell Biology International (2011) 35, 1147–1151 (Printed in Great Britain)
Effect of dexamethasone on human osteoblasts in culture: involvement of β1 integrin and integrin-linked kinase
Marcelo A. Naves*, Rosa M.R. Pereira†, Andréia N. Comodo*, Érika L.F.C. de Alvarenga*, Valéria F. Caparbo† and Vicente P.C. Teixeira*‡1
*Department of Pathology, Federal University of So Paulo, So Paulo, Brazil, †Division of Rheumatology, University of So Paulo School of Medicine, So Paulo, Brazil, and ‡Division of Nephrology, Department of Internal Medicine, Federal University of So Paulo, So Paulo, Brazil


Adhesive interactions play a critical role in cell biology, influencing vital processes from proliferation to cell death. Integrins regulate cell–ECM (extracellular matrix) adhesion and must associate with phosphorylating proteins such as ILK (integrin-linked kinase). Dysregulation of ILK expression is associated with anchorage-independent growth, cell survival and inhibition of apoptosis. Glucocorticoids influence differentiation and adhesion of osteoblasts and can affect bone protein synthesis. The objective of this study was to analyse the effect of DEX (dexamethasone) on the biology of osteoblasts, together with its influence on the expression of ILK and β1 integrin. For this, primary cultures of human osteoblasts were exposed to DEX at 10−9 M (physiological dose) and 10−6 M (pharmacological dose) for 24 and 48 h. Cell viability, apoptosis and cell adhesion were analysed, as well as protein expression of β1 integrin and ILK. It was observed that cell viability and adhesion were reduced in the cultures evaluated. In comparison with the control cultures, there was slightly less apoptosis in the cultures exposed to the physiological dose and considerably more apoptosis in those exposed to the pharmacological dose. In all treated cultures, protein expression of ILK was slightly higher than in the control cultures, whereas that of β1 integrin was significantly lower. Both proteins under study were co-localized at the cell periphery in all cultures. Our results suggest that DEX causes osteoblast anoikis, probably due to decreased β1 integrin expression, which might have had a direct influence upon ILK, reducing its activation and preventing it from playing its characteristic anti-apoptotic role.


Key words: adhesion ability, glucocorticoid, integrin-linked kinase (ILK), osteoblast, primary cell culture, β1 integrin

Abbreviations: DEX, dexamethasone, DMEM, Dulbecco's modified Eagle's medium, ECM, extracellular matrix, FBS, fetal bovine serum, GIOP, glucocorticoid-induced osteoporosis, ILK, integrin-linked kinase, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, TRITC, tetramethylrhodamine isothiocyanate

1To whom correspondence should be addressed (email vicente@nefro.epm.br).


1. Introduction

During the process of cell differentiation and maturation, bone cells can be influenced by a number of factors, such as glucocorticoid use (Canalis 1996). Glucocorticoids may influence several aspects of cell biology, such as survival, proliferation and apoptosis. Prolonged use of glucocorticoids can result in bone loss and GIOP (glucocorticoid-induced osteoporosis; Canalis et al., 2007). Glucocorticoids reduce the number, differentiation status and function of osteoblasts, as well as increase apoptosis in mature osteoblasts, which play a central role in the pathophysiology of GIOP (Pereira et al., 2001; Canalis et al., 2007; Mikami et al., 2008). A crucial aspect in cell biology is the interaction of cells with the ECM (extracellular matrix), which is modulated by membrane receptors such as integrins.

Integrins are transmembrane glycoproteins comprising two non-covalently associated subunits designated α and β. It is known that integrins can mediate transduction of signals that alter the genotypic and phenotypic characteristics of cells. This integrin-mediated modulation of cell functions is bidirectional, as intracellular signalling can regulate cell adhesion to the ECM and, similarly, signals emitted from ECM binding with extracellular integrin domains can modulate essential cell processes (Coppolino and Dedhar, 1999; Hood and Cheresh, 2002). Since integrins do not present enzymatic activity, they need to associate with other proteins in order to transmit signals originating from their activation (Coppolino and Dedhar, 1999). One of those proteins is ILK (integrin-linked kinase) that interacts with the cytoplasmic domain of integrin β-subunits (Hannigan et al., 1996).

ILK is a serine/threonine kinase that localizes in focal adhesion plaques, where it acts as a scaffold protein that amplifies signals from integrin activation. Since ILK modulates various essential cell functions, dysregulation of its expression is associated with cell-cycle progression, anchorage-independent growth, apoptosis inhibition, migration, mobility, invasion, tumour angiogenesis, epithelial–mesenchymal transdifferentiation and alternative activation of the Wnt signalling pathway (Hannigan et al., 2005). Grashoff et al. (2004) showed that altered expression of ILK induces abnormal cell adhesion to the ECM and prevents formation of focal adhesions, thereby altering cell adhesion and migration. Due to its influence on the cell processes mentioned above, ILK has been extensively studied, although there have been few studies addressing ILK in bone tissue.

In an attempt to increase knowledge of this issue, we focused the current investigation on the relationship between cells and the EMC. To that end, we evaluated the involvement of β1 integrin and ILK in the effect that the glucocorticoid DEX (dexamethasone) has on human osteoblasts in primary culture.

2. Materials and methods

2.1. Cell culture

The study protocol was approved by the Ethics in Research Committee of the Federal University of Sao Paulo. Human osteoblasts were obtained from primary culture as previously described (Caparbo et al., 2009). In brief, bone fragments were obtained from healthy individuals who were not using corticosteroids and had undergone orthopaedic surgery (donated by Dr Flavia Prada). Trabecular bone was cultured in DMEM (Dulbecco's modified Eagle's medium; Invitrogen Life Technologies, Carlsbad, CA, U.S.A.) supplemented with 25 mM Hepes, 500 units of penicillin G, 500 μg/ml of streptomycin and 3 μg/ml of amphotericin (Invitrogen) and 10% FBS (fetal bovine serum). Bone fragments were maintained in 15 ml of medium for 15 days, after which they were removed and discarded. Cells were allowed to grow until reaching confluence. After they were trypsinized, the cells were plated in six-well plates at 9×104 cells/cm2. After confluence had been reached, the phenotype was assessed by ALP (alkaline phosphatase) assay (Sigma–Aldrich, St Louis, MO, U.S.A.). In cell cultures that presented the osteoblastic phenotype, the medium was changed twice a week until confluence had been reached. In order to synchronize the largest possible number of cells in the G0 phase of the cell cycle, cells were cultured in DMEM with 3% FBS for 24 h prior to being exposed to DEX (Krek and DeCaprio, 1995; Merrill, 1998). DEX (Sigma–Aldrich) was added in two different doses (Lian et al., 1997; Mikami et al., 2008): a physiological dose (10−9 M) and a pharmacological dose (10−6 M) for 24 or 48 h. Cultures were therefore divided into six experimental groups: control, 24 h (untreated group); DEX (10−9 M), 24 h; DEX (10−6 M), 24 h; control, 48 h (untreated group); DEX (10−9 M), 48 h; DEX (10−6 M), 48 h.

2.2. Cell viability assay

Cell viability was quantified by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. After treatment, the cells were plated in 24-well plates and incubated for 2 h at 37°C with MTT reagent (Sigma–Aldrich). Formazan crystals, the product of the reaction between MTT reagent and viable cells, were dissolved in SDS/10% HCl solution. Absorbance was measured at 590 nm using a spectrophotometer (Labsystems Multiskan EX, Helsinki, Finland). Empty wells were used as blanks and were subtracted as background from each sample. Results were expressed as the mean absorbance obtained, higher absorbance translating to a greater number of viable cells.

2.3. Cell adhesion assay

The osteoblasts was measured adhesion as previously described (Forsprecher et al., 2009; Drury et al., 2010). After treatment, cells were trypsinized, seeded in 96-well plates (2×104 cells/well) and incubated for 6 h at 37°C in order to analyse the adherence capacity of osteoblasts. Attached cells were stained with a solution of 0.2% Crystal Violet in acetic acid for 15 min at room temperature. Stained cells were washed with water and were solubilized with 100 μl of methanol. Absorbance was measured at 590 nm. Results were expressed as the mean absorbance of the cultures, which is directly proportional to the number of cells adhering to the plate.

2.4. Apoptosis assay

Quantitative fluorescence of apoptotic bodies was performed using the DNA-intercalating dye propidium iodide as previously described (Krishan, 1975; Sträter et al., 1996; Claro et al., 2007). Cell suspensions were fixed in 50% ethanol and PBS for 1 h at 4°C. The samples (1×106 cells) were washed with PBS, centrifuged and the pellet was resuspended in PBS. Samples were then permeabilized using 0.01% saponin and incubated for 30 min with RNase A (4 mg/ml), after which propidium iodide (25 μg/ml) was added at room temperature. The samples were then analysed by FACSCalibur™ flow cytometry using the Cell Quest software (BD Biosciences, San Jose, CA, U.S.A.). Results were expressed as percentages of apoptotic cells in relation to the control values.

2.5. Immunofluorescence

Cells were fixed in 2% (w/v) paraformaldehyde (in PBS) and 4% sucrose for 10 min at room temperature, permeabilized in PBS with 0.5% Triton-X 100 for 10 min at 37°C and blocked with blocking buffer [2% FCS (fetal calf serum), 2% FSA (formaldehyde-treated serum albumin) and 0.2% fish gelatin]. Cells were incubated for 1 h with monoclonal anti-ILK antibody (1:50; Upstate Biotechnology, Lake Placid, NY, U.S.A.) or monoclonal anti-integrin β1 antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.). The subsequent steps were carried out for 1 h with TRITC (tetramethylrhodamine isothiocyanate) anti-mouse IgG or FITC anti-mouse IgG. For double staining, we used, in the same coverslip, a monoclonal anti-ILK antibody for 1 h and a TRITC anti-mouse IgG for 30 min. We then used a monoclonal anti-integrin β1 antibody for 1 h and a FITC anti-mouse IgG for 30 min. Slides were analysed under a confocal laser microscope (Bio-Rad, Hercules, CA, U.S.A.).

2.6. Western blotting

ILK and β1 integrin protein expression was determined by Western-blot analyses. Ten μg of protein was separated by SDS/PAGE under dissociating conditions on a 10% polyacrylamide slab gel using the Laemmli method (Laemmli, 1970). Proteins were stained with Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. α-Tubulin expression was used for quality control of the technique. Membrane bands were analysed using a Calibrated Imaging Densitometer and the Quantity One GS-710 software (Bio-Rad).

2.7. Statistical analysis

Values were expressed as means±S.E.M. To analyse the different variables, we used the unpaired Student's t test and the GraphPad Prism 5 software. The level of statistical significance was set at P<0.05.

3. Results

3.1. Effects of DEX on osteoblast viability

In all cultures treated with DEX for 24 h, viability was lower than that observed in the control cultures (Figure 1A). The cultures exposed to DEX for 48 h, at either dose, presented a statistically significant reduction in the number of viable cells in comparison with that observed for the control cultures. Regardless of the culture duration (24 or 48 h), there were no significant differences between the low-dose and high-dose cultures (Figure 1B).

3.2. Effects of DEX on cell adhesion

As shown in Figure 1(C), cell adhesion ability was significantly lower in osteoblasts exposed to DEX, at either dose, for 24 h compared with control cells. When we compared the doses per se, we found that cells exposed to higher dose presented lower cell adhesion ability in 24 h treatment. In osteoblasts exposed to DEX for 48 h, at either dose, the cell adhesion ability was also lower than in the control cells, and this difference was also statistically significant. In the cultures exposed to DEX for 48 h, the relationship between the two doses was similar to that observed in those exposed for 24 h: the number of adhered cells was inversely proportional to the dose, although the difference at 48 h was not statistically significant (Figure 1D).

3.3. Effects of DEX on osteoblast apoptosis

Figures 1(E) and 1(F) show the data related to the apoptotic assay in the cultures exposed to DEX for 24 and 48 h respectively. In both periods, the osteoblasts presented a similar response to DEX. The osteoblasts exposed to the lower dose of DEX presented a slight reduction in the percentage of apoptotic cells in comparison with the control cultures. On the other hand, there was a statistically significant difference between osteoblasts exposed to higher dose compared with control group.

3.4. Effects of DEX on expression of ILK and β1 integrin

In osteoblasts exposed to DEX for 24 or 48 h, at either dose, ILK expression was slightly higher than that seen in the respective control cultures, although the difference was not statistically significant (Figure 2). We found that β1 integrin expression was altered in osteoblasts exposed to DEX for 24 or 48 h. Cells exposed to either dose of DEX presented significantly lower β1 integrin expression than did those in the respective control cultures (Figure 3). Figure 4 shows typical immunofluorescence labelling for β1 integrin and ILK. As can be seen in Figure 4(A), β1 integrin is regularly distributed throughout the plasma membrane in thin lines suggestive of focal adhesion plaques. Figure 4(B) shows that ILK follows the pattern of β1 integrin, although it has a more coarse appearance, suggesting its role as a scaffold protein. There was no qualitative difference between the two proteins, in terms of their labelling, in any of experimental cultures, although there was a reduction in the quantity of cells in cultures exposed to DEX. In Figures 4(C-1)–4(C-3), both proteins are shown in the same cells. The juxtaposition of the images in Figure 4(C-3) reveals that β1 integrin and ILK are co-localized.

4. Discussion

Although it is known that treatment with DEX can alter the harmonic functioning of osteoblasts, there is no consensus regarding the exact effect that DEX has on bone tissue. In an attempt to increase knowledge of this issue, our investigation focused on the relationship between cells and the ECM.

In cells exposed to DEX, ILK expression was slightly increased, whereas there was substantial reduction in that of β1 integrin. This apparent paradox was also demonstrated by Weaver et al. (2008), who attributed it to a reduction in ILK activity, probably resulting from the loss of β1 integrin-mediated activation. The ability of β1 integrin to activate ILK was recently reported by Han et al. (2006). In their study, the authors detected gene and protein overexpression of β1 integrin, together with increased ILK expression and activity, and concluded in favour of a causal relationship between the two phenomena.

In the present study, cells exposed to DEX, at either dose, presented a drastic reduction in protein expression of β1 integrin. The reduction in β1 integrin expression was greatest in the cultures exposed for a longer period and to higher dose, suggesting that the effect of DEX on β1 integrin expression is dose- and time-dependent. Our results are consistent with studies showing that the use of glucocorticoids modulates β1 integrin expression (Gronowicz and McCarthy, 1995; Cooper, 2004). Although it has been established that glucocorticoids influence β1 integrin expression, the mechanism by which such inhibition occurs remains unclear.

Reduced integrin expression causes cell detachment, as observed in the present study, in which adhesion ability was significantly decreased in osteoblasts exposed to either DEX dose. In addition, cell detachment is an important stimulus for anoikis activation, which helps explain our findings (Frisch and Francis, 1994; Sträter et al., 1996; Frisch and Screaton, 2001; Marconi et al., 2007; Simpson et al., 2008). In cultures exposed to the higher dose of DEX, we found the apoptosis rate to be elevated at 24 h and at 48 h. In agreement with our findings, some studies have shown that high doses of DEX increase apoptosis rates in murine and human osteoblasts in primary and immortalized cultures (Conradie et al., 2007; Oshina et al., 2007; Espina et al., 2008).

The numbers of apoptotic osteoblasts were slightly lower in all of the cultures exposed to the lower dose of DEX than in the control cultures, reflecting a possible anti-apoptotic effect that has been attributed to reduced expression of pro-apoptotic genes such as Apaf-1 (apoptotic protease-activating factor 1), caspase 3 and caspase 9, together with increased expression of anti-apoptoic genes such as Bcl-xl (Pereira et al., 2001; Giner et al., 2007).

We found that osteoblast viability was reduced after treatment with DEX. Beloti and Rosa (2005) analysed the viability of human osteoblasts exposed to DEX in primary culture with similar results. However, in the present study, the difference between exposed and unexposed cultures reached statistical significance only in cultures exposed for 48 h, underscoring the hypothesis that DEX has a time-dependent effect on osteoblasts.

The intracellular localization of ILK in osteoblasts, here being determined by immunofluorescence for the first time, was proximal to the cell membrane, co-localized with β1-integrin, in accordance with results of previous studies (Han et al., 2006; Gkretsi et al. 2007).

Altogether, our results suggest that DEX causes greater cell death due to apoptosis as a result of lower cell adhesion, the ultimate consequence of reduced β1 integrin expression. Although our findings related to ILK participation in the effects of DEX were not conclusive, its influence cannot be completely ruled out, as it is closely related to integrin functions. Therefore further studies are necessary to clarify this issue as it is a promising source of future therapeutic strategies.

Author contribution

Marcelo A. Naves and Vicente P.C. Teixeira performed the conception and design of the study, analysis and interpretation of data and drafting of the article. Rosa M.R. Pereira did the analysis and interpretation of data and the critical revision of the article. Marcelo A. Naves, Andréia N. Comodo, Érika L.F.C. de Alvarenga and Valéria F. Caparbo did the collection and assembly of data.

Acknowledgement

We thank Dr Flavia Prada (Institute of Orthopedics and Traumatology, University of Sao Paulo, Sao Paulo, Brazil) for giving patients' bone fragments.

Funding

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo [grant number 07/54403-8].

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Received 8 October 2010/6 January 2011; accepted 3 June 2011

Published as Cell Biology International Immediate Publication 3 June 2011, doi:10.1042/CBI20100731


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ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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