Uncoupling protein 2 and 4 expression pattern during stem cell differentiation provides new insight into their putative function.

Apart from the first family member, uncoupling protein 1 (UCP1), the functions of other UCPs (UCP2-UCP5) are still unknown. In analyzing our own results and those previously published by others, we have assumed that UCP's cellular expression pattern coincides with a specific cell metabolism and changes if the latter is altered. To verify this hypothesis, we analyzed the expression of UCP1-5 in mouse embryonic stem cells before and after their differentiation to neurons. We have shown that only UCP2 is present in undifferentiated stem cells and it disappears simultaneously with the initiation of neuronal differentiation. In contrast, UCP4 is simultaneously up-regulated together with typical neuronal marker proteins TUJ-1 and NeuN during mESC differentiation in vitro as well as during murine brain development in vivo. Notably, several tested cell lines express UCP2, but not UCP4. In line with this finding, neuroblastoma cells that display metabolic features of tumor cells express UCP2, but not UCP4. UCP2's occurrence in cancer, immunological and stem cells indicates that UCP2 is present in cells with highly proliferative potential, which have a glycolytic type of metabolism as a common feature, whereas UCP4 is strongly associated with non-proliferative highly differentiated neuronal cells

Quantification of Uncoupling Protein 2 Reveals Its Main Expression in Immune Cells and Selective Up-Regulation during T-Cell Proliferation

<div><p>Uncoupling protein 2 (UCP2) is an inner mitochondrial membrane protein. Although the protein was discovered in 1997, its function and even its tissue distribution are still under debate. Here we present a quantitative analysis of mRNA and protein expression in various mice tissues, revealing that UCP2 is mainly expressed in organs and cells associated with the immune system. Although the UCP2 gene is present in the brain, as demonstrated using quantitative RT-PCR, the protein was not detectable in neurons under physiological conditions. Instead, we could detect UCP2 in microglia, which act in the immune defense of the central nervous system. In lymphocytes, activation led to a ten-fold increase of UCP2 protein expression simultaneously to the increase in levels of other mitochondrial proteins, whereas lymphocyte re-stimulation resulted in the selective increase of UCP2. The highest detected level of UCP2 expression in stimulated T-cells (0.54 ng/(µg total cellular protein)) was approximately 200 times lower than the level of UCP1 in brown adipose tissue from room temperature acclimated mice. Both the UCP2 expression pattern and the time course of up-regulation in stimulated T-cells imply UCP2’s involvement in the immune response, probably by controlling the metabolism during cell proliferation.</p> </div

Comparison of UCP2 mRNA and protein levels in different regions of the central nervous system.

<p>(A) UCP2 mRNA determination in 5 month old mice. UCP2 values are presented as ratios to the amount of GAPDH mRNA. Each data point represents the mean ± SD of six mice. (B) The best example for UCP2 protein detection in 50 µg total cellular protein of various regions of the central nervous system was performed in 5 month old mice.</p

Quantitative analysis of UCP2 expression in tissues using the recombinant proteins UCP2 and UCP1.

<p>(A) The comparison of UCP2 amounts in different tissue samples and isolated cells. UCP2 was calculated in ng of µg total protein in whole tissue (cell) lysate. Each data point represents the mean ± SE of six mice. (B) Representative Western Blot showing UCP2 quantification in thymus. (C) Representative Western Blot of UCP1 protein amount compared to the total cell protein in BAT.</p

Time course of UCP2 protein expression after unspecific stimulation (A), first re-stimulation (B) and second re-stimulation (C) of isolated T-cells.

<p>As mitochondrial markers antibodies against VDAC, Hsp60 and SDHA were used. Cell protein loading was controlled by detection of β-actin and GAPDH. The intensities of UCP2, VDAC, GAPDH, SDHA and Hsp60 were quantified as ratios to the intensity of β-actin. The graph demonstrated the percentage change in expression at 6 hours after the first re-stimulation (D). Each data point represents the mean ± SE of three different WB.</p

Lack of UCP4 expression in Dcx+/NeuN- neuroblasts in the adult subventricular zone (SVZ).

<p>A. Schematic drawing illustrates the localization of the SVZ of the lateral ventricle in adult mouse brain. B–C. Light microscopy analysis of the representative immunohistostained sample shows the distribution of UCP4- and Dcx-positive cells within the SVZ in 50 µm thick coronal sections of adult mouse brain. D. Representative CLSM images of UCP4 (green), Dcx (red) and NeuN (blue) stained with respective antibodies and visualized using Alexa 488, Alexa 594 and Alexa 633 fluorescent dyes.</p

UCP4 expression starts simultaneously with the expression of neuronal markers.

<p>A. UCP2, UCP4 and UCP5 mRNA levels during neuronal development analyzed by quantitative PCR. UCP mRNA levels in mouse head are shown as a ratio to GAPDH at embryonic day 12 (E12; inset) and as a ratio (UCP mRNA)/(GAPDH mRNA) at different days to (UCP mRNA)/(GAPDH mRNA) at E8. B. Representative Western blot indicates the simultaneous start of UCP4 protein expression with the expression of the neuronal marker TUJ-1. C-D. Representative Western blots demonstrate that UCP2 is not present at the protein level in the tested embryonic tissue (C) as well as in young postnatal neocortical brain tissue (NC) (D). Gels were loaded with 20 µg protein per lane. GAPDH, β-actin and VDAC were used as loading controls. At least 3 samples of pooled embryonic and postnatal tissue from at least 6 mice were analyzed at each condition (Experiments A–D).</p

Expression of neuronal/glial markers and UCP mRNA during neuronal differentiation.

<p>A. Representative Western blots show the time-dependent expression of neuronal (TUJ-1 for young and NF for adult neurons) and astrocyte (GFAP) markers during mESCs differentiation in culture. Gels were loaded with 20 µg protein per lane. B. Real-time PCR analysis of mESCs shows the amount of UCP2, UCP4 and UCP5 mRNA relative to mRNA amounts of the housekeeping gene GAPDH at different time points during neuronal differentiation. Each data point represents the mean value and SD of 3 independent differentiation experiments.</p

The neuroblastoma cell line N18TG2 expresses UCP2 but not UCP4.

<p>A. Representative Western blot analysis of UCP4 expression in the murine neuroblastoma cell line N18TG-2 and murine microglial cell line BV-2. Mouse brain tissue was used as a positive control for the antibody against UCP4. B. Representative Western blot analysis of UCP2 expression in the murine neuroblastoma cell line N18TG-2 and murine microglial cell line BV-2. Thymus of UCP2 knockout (KO) and wild type (wt) mice were used as negative and positive controls for the antibody directed against UCP4. Gels were loaded with 20 µg protein per lane. Cells from at least three different passages were analyzed in each experiment.</p