Tatjana Arsenijevic

Murine 3T3-L1 adipocyte cell differentiation model: validated reference genes for qPCR gene expression analysis.

Background Analysis of gene expression at the mRNA level, using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), mandatorily requires reference genes (RGs) as internal controls. However, increasing evidences have shown that RG expression may vary considerably under experimental conditions. We sought for an appropriate panel of RGs to be used in the 3T3-L1 cell line model during their terminal differentiation into adipocytes. To this end, the expression levels of a panel of seven widely used RG mRNAs were measured by qRT-PCR. The 7 RGs evaluated were ß-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl-transferase I (HPRT), ATP synthase H+ transporting mitochondrial F1 complex beta subunit (ATP-5b), tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide (Ywhaz), Non-POU-domain containing octamer binding protein (NoNo), and large ribosomal protein L13a (RPL). Methodology/Principal Findings Using three Excel applications, GeNorm, NormFinder and BestKeeper, we observed that the number and the stability of potential RGs vary significantly during differentiation of 3T3-L1 cells into adipocytes. mRNA expression analyses using qRT-PCR revealed that during the entire differentiation program, only NoNo expression is relatively stable. Moreover, the RG sets that were acceptably stable were different depending on the phase of the overall differentiation process (i.e. mitotic clonal expansion versus the terminal differentiation phase). RPL, ACTB, and Ywhaz, are suitable for terminal differentiation, whereas ATP-5b and HPRT, are suitable during mitotic clonal expansion. Conclusion Our results demonstrate that special attention must be given to the choice of suitable RGs during the various well defined phases of adipogenesis to ensure accurate data analysis and that the use of several RGs is absolutely required. Consequently, our data show for the first time, that during mitotic clonal expansion, the most suitable RGs are ATP-5b, NoNo and HPRT, while during terminal differentiation the most suitable RGs are, NoNo, RPL, ACTB and Ywhaz.

Distinct Specificities of pRb Phosphorylation by CDK4 Activated by Cyclin D1 or Cyclin D3: Differential Involvement in the Distinct Mitogenic Modes of Thyroid Epithelial Cells

Two distinct mitogenic modes coexist in the physiologically relevant model ofprimary cultures of dog thyroid epithelial cells. The differentiation-associated mitogenicstimulation by TSH and cAMP specifically requires the assembly and activation of cyclin D3-cyclin-dependent kinase (CDK)4 associated to p27kip1, while the dedifferentiatingproliferation induced by growth factors is associated with induction of cyclin D1. Here, wesuggest that the related CDK “inhibitors” p21cip1 and p27 are differentially utilized as positiveCDK4 regulators in these mitogenic stimulations. p21 was induced by EGF+serum, butrepressed by TSH, which, as previously shown, up-regulates p27. In response to EGF+serum,p21 supported the nuclear localization, phosphorylation and pRb-kinase activity of CDK4.Unexpectedly, partly different site-specificities of pRb-kinase activity, leading to similardifferences in the phosphorylation pattern of pRb in intact cells, were associated with cyclinD3-CDK4 bound to p27 in TSH-stimulated cells, or with CDK4 bound to p21 in growthfactor-stimulated cells. These differences were ascribed to the predominant association of thelatter complex to cyclin D1. Indeed, in different cell types and species, cyclin D1 varied fromcyclin D3 by more efficiently driving the phosphorylation of pRb at sites (Ser807/811 andThr826) required for its electrophoretic mobility shift. Therefore, different D-type cyclinscould differently impact some pRb functions, which should be considered not only in theunderstanding of the relationships between cell cycle and differentiation expression in thedistinct mitogenic modes of thyroid cells, but also in various development or differentiationmodels associated with dramatic switches in the expression of individual D-type cyclins.

G1/S Cyclins Interact with Regulatory Subunit of PKA via A-Kinase Anchoring Protein, AKAP95

The mechanisms by which cyclins promote mammalian cell cycle progression havebeen a topic of intense investigation over the last decade. We previously described aninteraction between D-type cyclins and A-kinase anchoring protein, AKAP95. Here, wedemonstrate that AKAP95 can also bind cyclin E1. Association between AKAP95 andcyclins is displaced by CDKs. We show that these G1/S cyclins can interact with RIIsubunit of PKAα through AKAP95. The presence of alternate complexes cyclin-CDKand cyclin D/E-AKAP95-PKA.RIIα suggest different roles of G1/S cyclins and a widerbiological importance of these interactions in cells.

Hyperosmotic stress induces cell cycle arrest in retinal pigmented epithelial cells

Osmotic changes occur in many tissues and profoundly influence cell function. Herein, we investigated the effect of hyperosmotic stress on retinal pigmented epithelial (RPE) cells using a microarray approach. Upon 4-h exposure to 100 mM NaCl or 200 mM sucrose, 79 genes were downregulated and 72 upregulated. Three gene ontology categories were significantly modulated: cell proliferation, transcription from RNA polymerase II promoter and response to abiotic stimulus. Fluorescent-activated cell sorting analysis further demonstrated that owing to hyperosmotic stimulation for 24 h, cell count and cell proliferation, as well as the percentage of cells in G0/G1 and S phases were significantly decreased, whereas the percentage of cells in G2/M phases increased, and apoptosis and necrosis remained unaffected. Accordingly, hyperosmotic conditions induced a decrease of cyclin B1 and D1 expression, and an activation of the p38 mitogen-activated protein kinase. In conclusion, our results demonstrate that hypertonic conditions profoundly affect RPE cell gene transcription regulating cell proliferation by downregulation cyclin D1 and cyclin B1 protein expression.

Pituitary Adenylate Cyclase Activating Peptide (PACAP) Participates in Adipogenesis by Activating ERK Signaling Pathway

Pituitary adenylate cyclase activating peptide (PACAP) belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. Its action can be mediated by three different receptor subtypes: PAC1, which has exclusive affinity for PACAP, and VPAC1 and VPAC2 which have equal affinity for PACAP and VIP. We showed that all three receptors are expressed in 3T3-L1 cells throughout their differentiation into adipocytes. We established the activity of these receptors by cAMP accumulation upon induction by PACAP. Together with insulin and dexamethasone, PACAP induced adipogenesis in 3T3-L1 cell line. PACAP increased cAMP production within 15 min upon stimulation and targeted the expression and phosphorylation of MAPK (ERK1/2), strengthened by the ERK1/2 phosphorylation being partially or completely abolished by different combinations of PACAP receptors antagonists. We therefore speculate that ERK1/2 activation is crucial for the activation of CCAAT/enhancer- binding protein β (C/EBPβ).

Involvement of JNK/NFκB Signaling Pathways in the Lipopolysaccharide-Induced Modulation of Aquaglyceroporin Expression in 3T3-L1 Cells Differentiated into Adipocytes

Aquaglyceroporins, belonging to the family of aquaporins (AQPs), are integral plasma membrane proteins permeable to water and glycerol that have emerged as key players in obesity. The aim of this study was to investigate the expression profile of AQPs in undifferentiated and differentiated 3T3-L1 cells and to investigate the changes in expression of aquaglyceroporins in 3T3-L1 cells differentiated into adipocytes and subjected to lipopolysaccharide (LPS) mimicking inflammation occurring during obesity. Furthermore, the study aimed at identifying the signaling cascade involved in the regulation of aquaglyceroporins expression upon LPS stimulation. 3T3-L1 cells were grown as undifferentiated cells (UDC; preadipocytes) or cells differentiated into adipocytes (DC, adipocytes). DC were incubated in the presence or absence of LPS with or without inhibitors of various protein kinases. AQPs mRNA expression levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). AQP1, AQP2, AQP3, AQP9 and AQP11 mRNA were expressed in both UDC and DC, whereas AQP4, AQP7 and AQP8 mRNA were expressed only in DC. In DC, LPS up-regulated AQP3 mRNA levels (p < 0.05) compared to control; these effects were inhibited by CLI095, SP600125 and BAY11-7082 (p < 0.05). LPS decreased both AQP7 and AQP11 mRNA levels (p < 0.01) in DC as compared to control; this decrease was inhibited by CLI095 and BAY11-7082 (p < 0.05) and additionally by SP00125 for AQP7 (p < 0.05). SB203580 had no effect on LPS-induced AQP3, AQP7 and AQP11 mRNA levels modulations. In conclusion, our results clearly show that many AQPs are expressed in murine 3T3-L1 adipocytes. Moreover, in DCs, LPS led to decreased AQP7 and AQP11 mRNA levels but to increased AQP3 mRNA levels, resulting from the Toll-like receptor 4 (TLR4)-induced activation of JNK and/or NFκB pathway.

Osmotic stress decreases aquaporin-4 expression in the human retinal pigment epithelial cell line, ARPE-19

The regulation of water movement is of utmost importance for normal retinal function. Under physiological conditions, water is transported, dependent on the osmotic gradient, through the retinal pigment epithelial cell layer from the subretinal space to the choroid. The osmotic gradient has been found to be modified in eye diseases, thus leading to water accumulation in the subretinal space and the sensory retina, and subsequently contributing to the formation of macular oedema. Understanding the regulation of aquaporin expression is therefore crucial. In this study, we investigated the effects of hyperosmolarity on aquaporin-4 (AQP4) protein expression in the human retinal pigment epithelial cell line, ARPE‑19. AQP4 expression was examined by PCR, western blot analysis and immunofluorescence. Ubiquitinylation was examined by immunoprecipitation. The results revealed that hyperosmotic stress rapidly decreased AQP4 expression in the ARPE-19 cells. The effect remained unmodified by lysosomal or mitogen-activated protein kinase inhibitors, but was reversed by proteasome inhibitors. However, no ubiquitinylation of AQP4 was detected. Our results suggest that hyperosmotic stress markedly reduces AQP4 expression possibly through a proteasome ubiquitinylation-independent pathway. This may represent an adaptation to hyperosmotic stress. The results presented in this study contribute to our understanding of the formation of macular oedema.

Forskolin Inhibits Lipopolysaccharide-Induced Modulation of MCP-1 and GPR120 in 3T3-L1 Adipocytes through an Inhibition of NFκB.

In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (IκBα). In DC, LPS increased MCP-1, TLR-4, and nuclear factor-κB1 (NFκB1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NFκB1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NFκB may be involved in the LPS-induced regulation of these genes.