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Bioscience Horizons 2008 1(1):1-8; doi:10.1093/biohorizons/hzn001
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© Oxford University Press 2008

Rosiglitazone-induced SERCA2b inhibition: implications for monocyte cytoskeletal remodelling and diabetic microangiopathy

Lisa Atkin*

University of Wales Institute Cardiff (UWIC), Cardiff, UK

* Corresponding author: No. 1, Mitre Court, Llandaff, Cardiff CF5 2EZ, UK. Tel: 0778 7768935. Email: l.j.atkin{at}uwic.ac.uk

Supervisor: Dr Richard Webb, University of Wales Institute Cardiff (UWIC), Cardiff, UK.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
In type 2 diabetes, exposure of monocytic cells to inflammatory stimuli can lead to aberrant cytoskeletal remodelling and contribute to the development of diabetic microangiopathy. Previous studies within our research group have demonstrated that Rosiglitazone significantly reduces formyl-methionyl-leucyl-proline (f-MLP)-induced actin polymerization in monocytes by increasing cytosolic calcium ion concentrations ([Ca2+]i) in both resting and f-MLP-stimulated cells. As the timescale of this effect resembled inhibition of SERCA2b, the ‘housekeeping’ Ca2+ pump enzyme that sequesters Ca2+ into the ER compensatory to non-specific leakage into the cytoplasm, the present study sought to test the hypothesis that Rosiglitazone-induced increases in [Ca2+]i are brought about by inhibition of SERCA2b, and also investigated how such changes are transduced into cytoskeletal remodelling.

Coupled-enzyme Ca2+-ATPase assays revealed a SERCA2b activity of 49 ± 5 nmol/mg/min in monocytic microsomes but pre-incubation with Rosiglitazone (20 µM; 30 min) induced statistically significant inhibition of this activity (P < 0.05).

Permeabilization of cultured monocytes in external solutions containing differing [Ca2+]free was used to manipulate [Ca2+]i, and F-actin content measured using flow cytometric analysis of FITC-phalloidin fluorescence. At [Ca2+]i >100 nM, a significant decrease in cells' F-actin content was observed (P < 0.05). In conditions of minimal [Ca2+]i, f-MLP exerted a polymerizing effect on the actin cytoskeleton; however, as [Ca2+]i was increased to >100 nM, this polymerizing effect was significantly reduced.

Finally, as a literature search identified the Ca2+-dependent actin-modulating protein gelsolin as a candidate for transducing changes in [Ca2+]i into actin cytoskeletal remodelling, western blot experiments using anti-gelsolin antibodies were used to detect ~88 kDa immunogenic bands in monocytic protein extracts, thus confirming the expression of gelsolin in monocytes.

In conclusion, the current study demonstrates the importance of [Ca2+]i in modulating actin cytoskeletal remodelling, and suggests that Rosiglitazone, via its ability to affect monocytic Ca2+ signalling processes, may confer a level of protection against the development of diabetic microangiopathy.

Key words: rosiglitazone, actin cytoskeleton, calcium, monocytes, gelsolin, diabetic microangiopathy


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Type 2 diabetes mellitus (T2D) is a common condition caused by the lack or diminished effectiveness of insulin, and is characterized by hyperglycaemia and other disruptions in metabolism. The condition is associated with a host of long-term complications that generally fall into two distinct categories, namely macrovascular and microvascular, which together constitute the major causes of morbidity and mortality amongst the diabetic population.1 In T2D, constant exposure of monocytic cells to inflammatory stimuli, such as the ligation of advanced glycation end products (AGEs) to their receptors on cell surface membranes, results in hyperactivation: a state characterized by increased polymerization of globular actin (G-actin) to the filamentous form (F-actin), which is accompanied by a concurrent reduction in cell deformability.2 Such aberrant monocytic cytoskeletal remodelling is thought to contribute to the development of diabetic microangiopathy and thus confer an increased risk for the progression of the microvascular complications of the disease, which include retinopathy, nephropathy, and neuropathy.3

Rosiglitazone is an anti-hyperglycaemic agent belonging to a group of compounds known as the thiazolidinediones (TZDs) that, owing to their role as ligands for the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-{gamma}), are used extensively to improve insulin sensitivity in individuals with T2D.4 Recently, our research group has demonstrated that in vitro treatment of human monocytic cells with Rosiglitazone significantly reduces the actin polymerization seen in response to the pro-inflammatory peptide formyl-methionyl-leucyl-proline (f-MLP).5 A similar decrease in f-MLP-induced actin polymerization was also observed in peripheral monocytes isolated from the blood of diabetic patients following a 3-month course of Rosiglitazone therapy (compared with peripheral monocytes from patients prescribed an alternative anti-diabetic drug, Gliclazide).6 Thus, these data suggest that in addition to its insulin-sensitizing effects, Rosiglitazone may possess the capacity to modulate the rigidity of the actin cytoskeleton of monocytes, and thus possibly confer a level of protection against the development of diabetic microangiopathy.

Since actin cytoskeletal remodelling is considered to be a Ca2+-dependent process,7, 8 it is interesting to note that Singh et al.5 also demonstrated that incubation with 20 µM Rosiglitazone (5–60 min) significantly increased cytosolic calcium ion concentrations ([Ca2+]i) in both resting and f-MLP-stimulated monocytic cells, an observation which, owing to the rapidity of the response, was deemed to represent a PPAR-{gamma}-independent non-genomic effect of the drug. Since the timescale of Rosiglitazone-induced increases in [Ca2+]i resembled those seen following inhibition of SERCA2b, the ‘housekeeping’ Ca2+ pump enzyme that sequesters Ca2+ into the ER compensatory to non-specific leakage into the cytoplasm,9 it was hypothesized that inhibition of this ATPase enzyme may be the mechanism responsible for Rosiglitazones's disruption of Ca2+ homeostasis. The current study sought to test this hypothesis by utilizing a coupled enzyme assay system to measure Ca2+-dependent ATP hydrolysis in monocytic microsomal fractions with or without pre-incubation with the putative SERCA2b inhibitor Rosiglitazone.

While the importance of changes in [Ca2+]i for facilitating remodelling of the leukocyte actin cytoskeleton has been widely reported,10, 11 it must be stressed that such a relationship remains somewhat controversial, with others suggesting that the process occurs independently of [Ca2+]i transients.12 Thus, a further aim of this study was to examine this relationship in more detail by using flow cytometric analyses to investigate the effect of varying [Ca2+]i on the F-actin content of FITC-phalloidin-stained electropermeabilized monocytes both prior and subsequent to f-MLP stimulation. In addition, since it has been widely acknowledged that actin cytoskeletal remodelling is accomplished via interactions with a multiplicity of accessory proteins,13, 14 monocytic cell protein lysates were subjected to western blot analyses so as to demonstrate expression in monocytes of the Ca2+-dependent protein gelsolin, a candidate protein for transducing changes in [Ca2+]i to rearrangements of the leukocyte actin cytoskeleton.

Finally, it should also be noted that, in the light of recent safety concerns linking treatment with Rosiglitazone to an increased risk of myocardial infarction,15, 16 a disease for which disrupted cellular Ca2+ signalling process are of undoubted importance,17 that data obtained in the present study may be of benefit in contributing to an assessment of the safety and efficacy of the drug in a clinical context.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Materials
All reagents were purchased from Sigma-Aldrich (Poole, UK) unless otherwise stated. Cell permeabilization reagents, protein assay reagents, FITC-phalloidin, and Fluo-3-AM were obtained from Harlan SERA-LAB (Loughborough, UK), Bio-Rad Laboratories (Herts, UK), Molecular Probes (Eugene, OR) and Calbiochem, EMD Biosciences (Darmstadt, Germany), respectively. Rosiglitazone was obtained from GlaxoSmithKline (Uxbridge, UK). Mouse monoclonal anti-gelsolin primary antibody was purchased from Abcam (Cambridge, UK) and HRP-labelled anti-mouse secondary antibody was purchased from New England Biolabs (Herts, UK). Cultured MM6 monocytic cells were obtained from the German Collection of Micro-Organisms and Cell Culture (Braunschwieg, Germany). Iterative CACONC software was a kind gift from Prof. A.G. Lee (Southampton University, UK).

Maintenance of cells in culture
Monocytic MM6 cells were cultured (37°C; 5% CO2) in RPMI 1640 medium supplemented with 10% (v/v) foetal calf serum, 1 mM non-essential amino acids, 2 mM L-glutamine and 1 mM sodium pyruvate and were sub-cultured when a density of 0.8–1.00 x 106 cells/ml was attained. Cells of >90% viability (as determined by Trypan blue exclusion) and passage number <30 were used in all instances, prior to harvesting of MM6 cells for further experiments.

Subcellular fractionation
MM6 microsomes were prepared by the method of Maruyama and MacLennan.18 Briefly, MM6 cells were homogenized, and whole-cell homogenates subjected to differential centrifugation so as to obtain different subcellular fractions, including microsomes. Previous analyses have demonstrated that MM6 microsomes prepared via this method co-purify with the ER marker protein calnexin.19

Ca2+-ATPase assays
A coupled enzyme assay in which generation of ADP was coupled to oxidation of NADH, and thus measured spectrophotometrically as a decrease in absorbance at 340 nm ({delta}A340 nm), was used to measure Ca2+-dependent ATP hydrolysis in monocytic microsomal fractions by the method of Webb et al.20 Briefly, microsomal samples (20–25 µg protein) were mixed and allowed to equilibrate for 15 min at 30°C in a reaction volume of 2.5 ml buffer (100 mM KCl, 40 mM Hepes, pH 7.2) containing ATP (2 mM), phosphoenolpyruvate (2.5 mM), NADH (0.25 mM), pyruvate kinase (7.5 IU) and lactate dehydrogenase (8 IU). Basal {delta}A340 nm readings were taken for 2 min using a Lambda25 spectrophotometer with UV-Winlab Software, following which 5 µl of the cation ionophore A23187 [GenBank] and 25 µl of 100 mM CaCl2 (determined by an iterative software package [CACONC] to give the desired free cation concentration of pCa4.82) were added to the cuvette, and {delta}A340 nm measured for a further 8 min. Corrected Ca2+-ATPase activities were determined by subtracting basal {delta}A340 nm readings from those recorded on the addition of calcium, and ATPase activity (expressed as nmol of ATP hydrolysed/mg of total protein/min) calculated using an extinction coefficient for NADH of 6220 M–1 cm–1. In some instances, microsomal samples were pre-incubated for 30 min at 30°C with 20 µM Rosiglitazone prior to addition to the reaction mixture.

Measurement of free Ca2+ concentrations ([Ca2+]free])
For the measurement of [Ca2+]free, 75 µl aliquots of permeabilization buffer (10 mM Hepes, 140 mM KCl, 1 mM MgCl2, 10 mM glucose, 1 mM ATP, pH 7.0) were transferred to fluorescence spectrophotometric cuvettes. 3 µM Fluo-3-AM was added to each cuvette, and fluorescence measured using a Perkin-Elmer Fluorescence Spectrophotometer with Perkin-Elmer Data Manager Software. Samples were excited at 506 nm and an emission scan performed between 510–600 nm; data were corrected for autofluorescence using measurements obtained from a solution of permeabilization buffer that had not been loaded with dye. To one cuvette, 10 µl of 100 mM CaCl2 (final concentration ~13 mM) was added to obtain a solution with maximal (i.e. saturating) Ca2+; to another 10 µl of 100 mM EGTA was added to obtain a solution with minimal Ca2+; while to the remaining cuvettes, 0.9 µl of 100 mM EGTA and aliquots of 100 mM CaCl2 predicted by CACONC to result in [Ca2+]free values of 2.88 µM, 0.75 µM, 0.50 µM, 0.25 µM and 83 nM were added. Fluorescence intensities at 524 nm for each sample (F), and for maximal Ca2+ (Fmax) and minimal Ca2+ (Fmin), were used to estimate absolute [Ca2+]free data in each case using the Grynkiewicz equation:21


Formula

(where ‘Kd’ = 400 nM, as previously reported22 for Fluo-3's dissociation constant with Ca2+). These observed data were then compared with predicted [Ca2+]free data, as calculated using CACONC.

Flow cytometry
The F-actin content of MM6 cells was determined using FITC-phalloidin, a fluorescently labelled derivative of the Amanita phalloides metabolite phalloidin, whose fluorescence has been reported to correlate with cellular F-actin content.23 Briefly, 107 cells were sedimented by centrifugation at 250g for 5 min, resuspended in 5 ml of ice-cold permeabilization buffer and pelleted (250g; 5 min). The resultant cell pellet was resuspended in ice-cold permeabilization buffer, and 300 µl aliquots of this suspension transferred to 0.4 cm3 electroporation cuvettes and subjected to an electrical pulse (200 V; 975 µF; t1/2, <30 ms) using a Bio-Rad Gene Pulser II (Bio-Rad Laboratories). Following electroporation, 0.9 µl of 100 mM EGTA and aliquots of 100 mM CaCl2, (predicted by CACONC to result in [Ca2+]free values of 2.88 µM, 0.75 µM, 0.25 µM and 83 nM) were added to 75 µl aliquots of the cell suspension, and cells allowed to equilibrate for 1 min at room temperature, before addition of FITC-phalloidin (1.65 µM) to each aliquot of cells, which were then incubated for 10 min at room temperature. Cells were then either fixed immediately by the addition of 10 µl of 37% phosphate-buffered formalin, or were fixed subsequent to incubation with 10 µl of 100 nM f-MLP for 5 min at 37°C. After fixation, cells were washed (x2) and resuspended in 0.5 ml of PBS, prior to analysis using a Beckman-Coulter Cytomecs-FC500-MPL FACS with FlowCytomix Pro1.0 software. The samples were excited by an argon laser at 488 nm and emission measured at 530 nm. Gating of cells was performed to exclude cellular debris, and the fluorescence intensity of each sample recorded as the gated x-mean. Autofluorescence was corrected for by analysis of fixed and unfixed electroporated cells in the absence of dye or f-MLP.

Western blot analysis
To confirm the expression of intracellular gelsolin in MM6 cells, total cell protein lysates were subjected to western blot analyses. 5 x 106 cells were sedimented by centrifugation at 300g for 10 min, washed (x3) in PBS, resuspended in 118 µl of lysis buffer (10 mM Tris–HCl pH 7.2, 100 mM NaCl, 2 mM EDTA, 0.5% w/v sodium deoxycholate, 1% v/v NonidetP40) and 12 µl of protease cocktail inhibitor, and maintained on ice for 30 min, during which time they were vortexed at three separate intervals. Samples were sonicated on ice for 10 s and centrifuged at 8000g for 5 min at 4°C; supernatants were then subjected to Bio-Rad DC protein assays in order to determine the protein concentration of cell lysates. Western blot analyses were performed using 100 µg of protein in each instance: after electrophoresis (10% SDS–PAGE), samples were transferred onto nitrocellulose membranes, which were incubated with blocking buffer (Tris-buffered saline containing 0.1% Tween 20 [TBS-T] and 4% skimmed milk) for 60 min at room temperature before probing with mouse monoclonal anti-gelsolin primary antibody (18 h; 1:1000 dilution in blocking buffer), followed by HRP-labelled anti-mouse secondary antibody (2 h; 1:2000 dilution in blocking buffer), with washing (3 x 5 min; TBS-T) between each step. Immunogenic protein was detected by incubation with 1 ml West Pico Dura luminol/peroxide substrate (1:1; 5 min), and visualization of ECL-enhanced chemiluminescence using a UVP Gel Documentation system.

Statistical analysis
The data presented in Fig. 2 were analysed by analysis of variance (ANOVA), while the data presented in Fig. 3, the Ca2+-ATPase data and the data from the experiment designed to investigate the effect of f-MLP on the F-actin content of cells were analysed by two-sample t-tests. Significance levels were set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Ca2+-ATPase activity
At a [Ca2+]free of pCa4.82 (which has previously been identified as the [Ca2+]free at which SERCA2b exhibits optimal Ca2+-ATPase activity24), a Ca2+-dependent ATPase activity of 49 ± 5 nmol/mg/min was identified in MM6 microsomal fractions. This activity was abolished (0 ± 8 nmol/mg/min; P < 0.05; n = 3) following pre-incubation (30 min; 30°C) with 20 µM Rosiglitazone.

Measurement of [Ca2+]free
While there were some discrepancies between the [Ca2+]free values predicted using CACONC and the [Ca2+]free values observed using fluorescence spectrophotometric data (Fig. 1), the ‘expected’ and ‘observed’ [Ca2+]free values coincided in all cases where physiologically relevant [Ca2+]free values (i.e. <0.5 µM25) were used (Table 1). Thus, it was confirmed that the aliquots of CaCl2 predicted by CACONC to result in the desired [Ca2+]free values in our experimental system did indeed produce [Ca2+]free of approximately 2.88 µM, 0.75 µM, 0.25 µM and 83 nM. As electroporation produces cells whose ‘leaky’ plasma membranes allow intracellular access to biological molecules,6 it was assumed that in subsequent experiments, addition of CaCl2 to the external solution resulted in diffusion of Ca2+ to the cytoplasm of permeabilized cells—thus, [Ca2+]free in the external permeabilization buffer = [Ca2+]i within permeabilized cells.


Figure 1
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  		Figure 1. Emission spectra illustrating the relationship between [Ca2+]free and fluorescence intensity of the Ca2+ indicator dye Fluo-3 ({lambda}ex = 506 nm; {lambda}em = 510–600 nm). Spectra (in order of decreasing fluorescence intensity) correspond to Fmax, [Ca2+]free = 3.25 µM, [Ca2+]free = 1.85 µM, [Ca2+]free = 0.54 µM, [Ca2+]free = 0.25 µM, [Ca2+]free = 97 nM and Fmin.

 


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  		Table 1. Free [Ca2+] concentrations, calculated using two independent methods: iterative CACONC software and conversion of Fluo-3 fluorescence to [Ca2+]free values

 
Effect of varying [Ca2+]i on F-actin content
To investigate the effect of [Ca2+]i on the polymerization of the actin cytoskeleton, MM6 cells were permeabilized by electroporation, allowed to equilibrate in buffer containing FITC-phalloidin and appropriate amounts of CaCl2 to result in the desired [Ca2+]i values, following which F-actin content was measured using flow cytometric analysis of FITC-phalloidin fluorescence. Increasing [Ca2+]i above resting levels (>100 nM) resulted in a substantial decrease in the F-actin content of cells (Fig. 2). The effect of increasing [Ca2+]i on the polymerization of the actin cytoskeleton was observed to be greatest between 0.25 µM and 0.75 µM, an effect consistent with both the physiological range in which transient increases in [Ca2+]i result in cellular activation, and with those [Ca2+]i values previously reported by Yin, Zaner and Stossel26 to result in activation of gelsolin. Furthermore, the depolymerizing effect of increasing [Ca2+]i was seen to plateau at supraphysiological [Ca2+]i values. Thus, these data indicate that increasing [Ca2+]i exerts a depolymerizing effect on the actin cytoskeleton (P < 0.05; ANOVA; n = 3).


Figure 2
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  		Figure 2. Dose–response curve illustrating depolymerization of the actin cytoskeleton, as measured using FACS analysis of FITC-phalloidin fluorescence, of electroporated MM6 cells bathed in external solutions of the indicated [Ca2+]free (P < 0.05; ANOVA). Each value represents the mean ± SEM of three determinations.

 
Effect of f-MLP on F-actin content
A ‘positive control’ experiment was carried out to confirm that, in conditions of minimal [Ca2+]i, f-MLP stimulation (100 nM; 5 min) exerted a polymerizing effect on the actin cytoskeleton. Comparison of the FITC-phalloidin fluorescence intensities of MM6 cells in the absence or presence of f-MLP stimulation demonstrated that f-MLP brought about a significant increase (~1.7 fold) in fluorescence intensity (from 7.67 ± 1.82 to 13.01 ± 2.65 Relative Fluorescent Units; P < 0.05; n = 6) and thus F-actin content.

Effect of varying [Ca2+]i on f-MLP-induced actin polymerization
To investigate the effect of [Ca2+]i on f-MLP-induced actin polymerization, MM6 cells were permeabilized by electroporation, allowed to equilibrate in buffer containing FITC-phalloidin and appropriate amounts of CaCl2 to result in the desired [Ca2+]i values, following which cells were exposed to f-MLP (100 nM; 5 min) and F-actin content measured. Exposure to f-MLP at all [Ca2+]i values investigated was shown to be associated with non-significant increases in the F-actin content of cells above basal (Fig. 3; P > 0.05 in all cases; n = 4; values for basal F-actin levels were derived from preliminary experiments performed at each [Ca2+]i value in the absence of f-MLP). In all instances, this increase was observed to be less than that apparent in the earlier ‘positive control’ experiment (~170%, shown as the first data point in Fig. 3). Thus, although it is somewhat difficult to establish a clear trend, it would appear that as the level of [Ca2+]i is increased above resting levels, the polymerizing effect of f-MLP diminishes so that it no longer attains statistical significance. It is therefore possible to state that, in our experimental system, the depolymerizing effect of Ca2+ may override the polymerizing action of f-MLP.


Figure 3
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  		Figure 3. Dose–response curve illustrating Ca2+-dependent amelioration of f-MLP (100 nM; 5 min)-induced actin polymerization, as measured using FACS analysis of FITC-phalloidin fluorescence, in electroporated MM6 cells bathed in external solutions of indicated [Ca2+]free (P > 0.05 vs. basal F-actin content at each [Ca2+]free value). Each value represents mean ± SEM of four determinations expressed as a percentage of the F-actin content of cells at each [Ca2+]free value in the absence of f-MLP.

 
Gelsolin expression in MM6 cells
In western blot experiments (Fig. 4), gelsolin was detected in six separate MM6 total cell protein lysates as bands with a mean molecular weight of ~88 kDa, which is in good agreement with that previously reported for gelsolin by Yin and Stossel.27


Figure 4
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  		Figure 4. Western blot analysis using anti-gelsolin mouse monoclonal primary antibodies and HRP-labelled anti-mouse secondary antibodies to detect the presence of gelsolin in MM6 total cell protein lysates (lanes 2–7). Lane 1 represents molecular weight markers. Arrow indicates mean molecular weight (MW) of immunogenic bands.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
This study has demonstrated that pre-incubation of monocytic microsomal fractions with 20 µM Rosiglitazone abolishes Ca2+ ATPase activity. Since studies within our research group have confirmed the expression of SERCA2b in MM6 microsomal membranes,28 it can be concluded that the Rosiglitazone-induced increases in [Ca2+]i seen in monocytic cells, as previously reported by Singh et al., 5 be attributable to inhibition of this ER-resident Ca2+ pump enzyme. It is likely that the capacity of Rosiglitazone to inhibit the activity of SERCA2b represents a non-genomic effect of the drug, since inhibitory activity was evident following relatively short periods of incubation (30 min) and was observed in microsomal fractions that lack nuclear proteins such as PPAR-{gamma}.18

When considering the relevance of these data in a clinical context, it is important to be aware that the concentration of Rosiglitazone selected in the current study is not strictly applicable to the in vivo state where the peak plasma concentration of the drug is estimated to be ~0.8 µM.29 However, subsequent studies on MM6 cells within our research group have recently demonstrated that statistically significant inhibition of Ca2+ ATPase activity remains evident at pharmacologically relevant concentrations.28

As described earlier, SERCA2b performs a ‘housekeeping’ role in sequestering Ca2+ into the ER compensatory to non-specific leakage into the cytoplasm;9 thus, a consequence of Rosiglitazone-induced SERCA2b inhibition would be leakage of Ca2+ from the ER, and elevated [Ca2+]i, as previously seen in our laboratory.5 Because of the importance of aberrant monocyte cytoskeletal remodelling to the development of diabetic microangiopathy,3 our subsequent experiments aimed to elucidate the effects of increased [Ca2+]i on monocyte actin polymerization.

Although there has been some previous debate as to the Ca2+ dependence of leukocyte actin cytoskeletal remodelling,10–12 the data obtained in the current study suggest that changes in [Ca2+]i do indeed modulate actin cytoskeletal remodelling, either in the absence (Fig. 2) or the presence (Fig. 3) of activating cell stimuli. Since the expression of gelsolin—as seen here in MM6 cells (Fig. 4)—has been reported in a wide variety of cell types,13 and as the actin depolymerizing activity of gelsolin is Ca2+-dependent,30 it seems reasonable to tentatively conclude that decreased F-actin content following elevation of [Ca2+]i is likely to be attributable to actin filament severing by gelsolin, rather than being due to any direct action of the Ca2+ion on actin. Clearly, however, further work is required to confirm this.

In line with previous experimental findings by Downey et al., 31 our data have shown that as [Ca2+]i is increased above resting levels, the polymerizing effect of f-MLP diminishes so that it no longer attains statistical significance (Fig. 3). f-MLP was utilized in this investigation as a surface receptor agonist with which to mimic the inflammatory stimuli, such as AGEs, to which monocytic cells are exposed in T2D. Therefore, our data suggest that Rosiglitazone, via its ability to modulate monocytic Ca2+ signalling processes, may serve to counter the promotion of excessive actin polymerization in monocytes following exposure to inflammatory stimuli, as seen in the diabetic milieu.32

Thus, our data suggest that treatment of monocytic cells with pharmacologically relevant concentrations of Rosiglitazone leads to inhibition of SERCA2b resulting in elevations in [Ca2+]i. Such perturbations in [Ca2+]i subsequently facilitate actin cytoskeletal depolymerization, possibly via activation of the Ca2+-dependent actin-modulating protein gelsolin, which ultimately results in increased deformability of monocytes, and as such may confer a level of protection against the development of diabetic microangiopathy. The observation that the depolymerizing effect of Ca2+ appears to override the polymerizing action of activating cell stimuli provides a sound rationale for the potentially beneficial effects of Rosiglitazone on modulating the actin cytoskeleton of hyperactivated monocytes. However, further investigations, perhaps involving in vitro studies using AGEs as inflammatory stimuli of direct relevance to T2D, and—in the long-term—patient studies would be recommended to test the validity of these initial hypotheses.

Finally, given the current controversy linking treatment with Rosiglitazone to an increased risk of myocardial infarction and of death from cardiovascular causes,15, 16 it is worth considering the relevance of the data obtained in the present study to these recent clinical concerns. In cardiomyocytes, contraction is triggered by transient increases in [Ca2+]i, while relaxation is mediated by the rapid sequestration of cytosolic Ca2+ predominantly through SERCA2a (the alternatively-spliced isoform of SERCA2b24), the activity of which is modulated by an accessory polypeptide, phospholamban.33 Studies involving genetically engineered mouse models have demonstrated that the hearts of SERCA2a heterozygous mutants exhibited decreased cardiac function, while models overexpressing a mutant form of phospholamban displayed depressed contractile parameters, which ultimately led to heart failure and death.34 Such data highlight the importance of Ca2+-regulatory proteins to the correct functioning of the heart and suggest that while the capacity for Rosiglitazone to inhibit SERCA2 may be clinically beneficial in the context of hyperactivated diabetic monocytes, it has the potential to be deleterious in the context of the cardiomyocyte. It must be stressed, however, that it remains to be seen whether the mechanisms via which Rosiglitazone disrupts Ca2+ homeostasis in monocytes also operate in other cell types, and thus further investigation is recommended in order for a clearer picture to emerge.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
This project was funded by the UWIC Centre for Biomedical Sciences.


   Acknowledgements
 
I acknowledge the guidance and support of my supervisor, Dr Richard Webb, and members of his research group.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

  1. Massi-Benedetti M. The cost of diabetes type II in Europe. Diabetologia (2002) 45:S1–S4.[CrossRef][Web of Science][Medline]
  2. Schroder S, Palinski W, Schmid-Schonbein GW. Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy. Am J Pathol (1991) 139:81–100.[Abstract]
  3. Miyamoto K, Ogura Y, Kenmochi S, et al. Role of leukocytes in diabetic microcirculatory disturbances. Microvasc Res (1997) 54:43–48.[CrossRef][Web of Science][Medline]
  4. Smith SA. Central role of the adipocyte in the insulin-sensitising and cardiovascular risk modifying actions of the thiazolidinediones. Biochimie (2003) 12:1219–1230.
  5. Singh N, Webb R, Adams R, et al. The PPAR-{gamma} activator, Rosiglitazone, inhibits actin polymerisation in monocytes: involvement of Akt and intracellular calcium. Biochem Biophys Res Commun (2005) 333:455–462.[CrossRef][Web of Science][Medline]
  6. Singh N. Rosiglitazone-induced PPAR-{gamma}-dependent and -independent anti-inflammatory effects in monocytic cells. (2007) UWIC. PhD thesis.
  7. Yin HL. Gelsolin: calcium-and polyphosphoinositide-regulated actin-modulating protein. BioEssays (1987) 7:176–179.[CrossRef][Web of Science][Medline]
  8. Matsudaira P, Janmey P. Pieces in the actin-severing protein puzzle. Cell (1988) 54:139–140.[CrossRef][Web of Science][Medline]
  9. John LM, Lechleiter JD, Camacho P. Differential modulation of SERCA2 isoforms by careticulin. J Cell Biol (1998) 142:963–973.[Abstract/Free Full Text]
  10. Maxfield FR. Regulation of leukocyte locomotion by Ca2+. Trends Cell Biol (1993) 11:386–391.
  11. May RC, Machesky LM. Phagocytosis and the actin cytoskeleton. J Cell Sci (2001) 114:1061–1077.[Abstract]
  12. Al-Mohanna FA, Hallett MB. Actin polymerization in neutrophils is triggered without a requirement for a rise in cytoplasmic Ca2+. Biochem J (1990) 266:669–674.[Web of Science][Medline]
  13. Yin HL, Albrecht JH, Fattoum A. Identification of gelsolin, a Ca2+-dependent regulatory protein of actin gel-sol transformation, and its intracellular distribution in a variety of cells and tissues. J Cell Biol (1981) 91:901–906.[Abstract/Free Full Text]
  14. Silacci P, Mazzolai L, Gauci C, et al. Gelsolin superfamily proteins: key regulators of cellular functions. Cell Mol Life Sci (2004) 61:2614–2623.[CrossRef][Web of Science][Medline]
  15. Home PD, Pocock SJ, Beck-Nielsen H, et al. Rosiglitazone evaluated for cardiovascular outcomes—an interim analysis. N Engl J Med (2007) 357:28–38.[Abstract/Free Full Text]
  16. Nissen SE, Wolski K. Effect of Rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes. N Engl J Med (2007) 356:2457–2471.[Abstract/Free Full Text]
  17. Bers DM. Cardiac excitation-contraction coupling. Nature (2002) 415:198–205.[CrossRef][Medline]
  18. Maruyama K, MacLennan DH. Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells. Proc Natl Acad Sci USA (1988) 85:3314–3318.[Abstract/Free Full Text]
  19. Ulug P. Characterisation of a putative Ca2+/Mn2+ signalling system located in the ER and Golgi of eukaryotic cells. (2004) UWIC. MSc dissertation.
  20. Webb RJ, Khan YM, East JM, et al. The importance of carboxyl groups on the lumenal side of the membrane for the function of the Ca2+-ATPase of sarcoplasmic reticulum. J Biol Chem (2000) 275:977–982.[Abstract/Free Full Text]
  21. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem (1985) 260:3440–3450.[Abstract/Free Full Text]
  22. Merritt JE, McCarthy SA, Davies MPA, et al. Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Biochem J (1990) 269:513–519.[Web of Science][Medline]
  23. Howard TH, Meyer WH. Chemotactic peptide modulation of actin assembly and locomotion in neutrophils. J Cell Biol (1984) 98:1265–1271.[Abstract/Free Full Text]
  24. Lytton J, Westlin M, Burk SE, et al. Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps. J Biol Chem (1992) 267:14483–14489.[Abstract/Free Full Text]
  25. Advani A, Marshall SM, Thomas TH. Impaired neutrophil store-mediated calcium entry in type 2 diabetes. Eur J Clin Invest (2004) 34:43–49.[CrossRef][Web of Science][Medline]
  26. Yin HL, Zaner KS, Stossel TP. Ca2+ control of actin gelation. J Biol Chem (1980) 255:9494–9500.[Abstract/Free Full Text]
  27. Yin HL, Stossel TP. Purification and structural properties of gelsolin, a Ca2+-activated regulatory protein of macrophages. J Biol Chem (1980) 255:9490–9493.[Abstract/Free Full Text]
  28. Caddy J, Singh N, Atkin L, et al. Rosiglitazone transiently disturbs calcium homeostasis in monocytic cells. Biochem Biophys Res Comm (2008) 366:149–155.[CrossRef][Web of Science][Medline]
  29. Niemi M, Backman JT, Granfors M, et al. Gemfibrozil considerably increases the plasma concentrations of Rosiglitazone. Diabetologia (2003) 46:1319–1323.[CrossRef][Web of Science][Medline]
  30. Yin HL, Stossel TP. Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein. Nature (1979) 281:583–586.[CrossRef][Medline]
  31. Downey GP, Chan CK, Trudel S, et al. Actin assembly in electropermeabilized neutrophils: role of intracellular calcium. J Cell Biol (1990) 110:1975–1982.[Abstract/Free Full Text]
  32. Cipollone F, Iezzi A, Fazia M, et al. The receptor RAGE as a progression factor amplifying arachidonate-dependent inflammatory and proteolytic response in human atherosclerotic plaques. Circulation (2003) 108:1070–1077.[Abstract/Free Full Text]
  33. Mørk HK, Sjaastad I, Sande JB, et al. Increased cardiomyocyte function and Ca2+ transients in mice during early congestive heart failure. J Mol Cell Cardiol (2007) 43:177–186.[CrossRef][Web of Science][Medline]
  34. Chu G, Haghighi K, Kranias EG. From mouse to man: understanding heart failure through genetically altered mouse models. J Card Fail (2002) 8:S432–S449.[CrossRef][Web of Science][Medline]
Submitted on 24 September 2007; accepted on 17 December 2007


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