Adaptations of the 3T3-L1 adipocyte lipidome to defective ether lipid catabolism upon Agmo knockdown.

Little is known about the physiological role of alkylglycerol monooxygenase (AGMO), the only enzyme capable of cleaving the 1-O-alkyl ether bond of ether lipids. Expression and enzymatic activity of this enzyme can be detected in a variety of tissues including adipose tissue. This labile lipolytic membrane-bound protein uses tetrahydrobiopterin as a cofactor, and mice with reduced tetrahydrobiopterin levels have alterations in body fat distribution and blood lipid concentrations. In addition, manipulation of AGMO in macrophages led to significant changes in the cellular lipidome, and alkylglycerolipids, the preferred substrates of AGMO, were shown to accumulate in mature adipocytes. Here, we investigated the roles of AGMO in lipid metabolism by studying 3T3-L1 adipogenesis. AGMO activity was induced over 11 days using an adipocyte differentiation protocol. We show that RNA interference-mediated knockdown of AGMO did not interfere with adipocyte differentiation or affect lipid droplet formation. Furthermore, lipidomics revealed that plasmalogen phospholipids were preferentially accumulated upon Agmo knockdown, and a significant shift toward longer and more polyunsaturated acyl side chains of diacylglycerols and triacylglycerols could be detected by mass spectrometry. Our results indicate that alkylglycerol catabolism has an influence not only on ether-linked species but also on the degree of unsaturation in the massive amounts of triacylglycerols formed during in vitro 3T3-L1 adipocyte differentiation.

Carbonic anhydrase III protects osteocytes from oxidative stress.

Osteocytes are master orchestrators of bone remodeling; they control osteoblast and osteoclast activities both directly via cell-to-cell communication and indirectly via secreted factors, and they are the main postnatal source of sclerostin and RANKL (receptor activator of NF-kB ligand), two regulators of osteoblast and osteoclast function. Despite progress in understanding osteocyte biology and function, much remains to be elucidated. Recently developed osteocytic cell lines-together with new genome editing tools-has allowed a closer look at the biology and molecular makeup of these cells. By using single-cell cloning, we identified genes that are associated with high Sost/sclerostin expression and analyzed their regulation and function. Unbiased transcriptome analysis of high- vs. low-Sost/sclerostin-expressing cells identified known and novel genes. Dmp1 (dentin matrix protein 1), Dkk1 (Dickkopf WNT signaling pathway inhibitor 1), and Phex were among the most up-regulated known genes, whereas Srpx2, Cd200, and carbonic anhydrase III (CAIII) were identified as novel markers of differentiated osteocytes. Aspn, Enpp2, Robo2, Nov, and Serpina3g were among the transcripts that were most significantly suppressed in high-Sost cells. Considering that CAII was recently identified as being regulated by Sost/sclerostin and capable of controlling mineral homeostasis, we focused our attention on CAIII. Here, we report that CAIII is highly expressed in osteocytes, is regulated by parathyroid hormone both in vitro and in vivo, and protects osteocytes from oxidative stress.-Shi, C., Uda, Y., Dedic, C., Azab, E., Sun, N., Hussein, A. I., Petty, C. A., Fulzele, K., Mitterberger-Vogt, M. C., Zwerschke, W., Pereira, R., Wang, K., Divieti Pajevic, P. Carbonic anhydrase III protects osteocytes from oxidative stress.

Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells.

BACKGROUND: The proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a gene of interest. This approach requires constitutively expressed reference genes for normalization to counteract inter-sample variations due to differences in RNA quality and quantity. Thus, a careful validation of quantitative RT-PCR reference genes is needed to accurately measure fluctuations in the expression of genes. Here, we evaluated candidate reference genes applicable for quantitative RT-PCR analysis of gene expression during proliferation and adipogenesis of human ASCs with the immunophenotype DLK1+/CD34+/CD90+/CD105+/CD45-/CD31-. METHODS: We evaluated the applicability of 10 candidate reference genes (GAPDH, TBP, RPS18, EF1A, TFRC, GUSB, PSMD5, CCNA2, LMNA and MRPL19) using NormFinder, geNorm and BestKeeper software. RESULTS: The results indicate that EF1A and MRPL19 are the most reliable reference genes for quantitative RT-PCR analysis of proliferating ASCs. PSMD5 serves as the most reliable endogenous control in adipogenesis. CCNA2 and LMNA were among the least consistent genes. CONCLUSIONS: Applying these findings for future gene expression analyses will help elucidate ASC biology.

Activators and stimulators of soluble guanylate cyclase counteract myofibroblast differentiation of prostatic and dermal stromal cells.

BACKGROUND: Fibrotic diseases encompass numerous systemic and organ-specific disorders characterized by the development and persistence of myofibroblasts. TGFß1 is considered the key inducer of fibrosis and drives myofibroblast differentiation in cells of diverse histological origin by a pro-oxidant shift in redox homeostasis associated with decreased nitric oxide (NO)/cGMP signaling. Thus, enhancement of NO/cGMP represents a potential therapeutic strategy to target myofibroblast activation and therefore fibrosis. METHODS: Myofibroblast differentiation was induced by TGFß1 in human primary prostatic (PrSCs) and normal dermal stromal cells (NDSCs) and monitored by α smooth muscle cell actin (SMA) and IGF binding protein 3 (IGFBP3) mRNA and protein levels. The potential of enhanced cGMP production by the sGC stimulator BAY 41-2272 or the sGC activator BAY 60-2770 to inhibit and revert myofibroblast differentiation in vitro was analyzed. Moreover, potential synergisms of BAY 41-2272 or BAY 60-2770 and inhibition of cGMP degradation by the PDE5 inhibitor vardenafil were investigated. RESULTS: BAY 41-2272 and BAY 60-2770 at doses of 30µM significantly inhibited induction of SMA and IGFBP3 levels in PrSCs and reduced myofibroblast marker levels in TGFß1-predifferentiated cells. At lower concentrations (3 and 10µM) only BAY 41-2272 but not BAY 60-2770 significantly inhibited and reverted myofibroblast differentiation. In NDSCs both substances significantly inhibited differentiation at all concentrations tested. Attenuation of SMA expression was more pronounced in NDSCs whereas reduction of IGFBP3 levels by BAY 41-2272 appeared more efficient in PrSCs. Moreover, administration of BAY 41-2272 or BAY 60-2770 enhanced the efficiency of the PDE5 inhibitor vardenafil to inhibit and revert myofibroblast differentiation in vitro. CONCLUSIONS: Increase of cGMP by sGC stimulation/activation significantly inhibited and reverted myofibroblast differentiation. This effect was even more pronounced when a combination treatment with a PDE5 inhibitor was applied. Thus, enhancement of NO/cGMP-signaling by sGC stimulation/activation is a promising strategy for the treatment of fibrotic diseases. Whereas, in NDSCs BAY 60-2770 and BAY 41-2272 exerted similar effects on myofibroblast differentiation, higher potency of BAY 41-2272 was observed in PrSCs, indicating phenotypical differences between fibroblasts form different organs that should be taken into account in the search for antifibrotic therapies.

Depletion of the cdk inhibitor p16INK4a differentially affects proliferation of established cervical carcinoma cells.

UNLABELLED: Infections with high-risk human papillomaviruses (hrHPV) contribute to cervical carcinoma. The cdk inhibitor and tumor suppressor p16INK4A is consistently upregulated in cervical carcinoma cells for reasons that are poorly understood. We report here that downregulation of p16INK4A gene expression in three different cervical carcinoma cell lines reduced expression of the E7 oncogene, suggesting a positive feedback loop involving E7 and p16INK4A. p16INK4A depletion induced cellular senescence in HeLa but not CaSki and MS-751 cervical carcinoma cells. IMPORTANCE: This study demonstrates that the cdk inhibitor p16INK4A, frequently used as surrogate marker for transforming infections by human papillomaviruses of the high-risk group, is required for high-level expression of the E7 oncoproteins of HPV-16, HPV-18, and HPV-45 in cervical carcinoma cells. It is also demonstrated that depletion of p16INK4A induces senescence in HeLa but not CaSki or MS-751 cervical carcinoma cells.

Analysis of human papillomavirus E7 protein status in C-33A cervical cancer cells.

High-risk human papillomaviruses (HPV) are the main etiologic factor for the development of cervical cancer. Infections by these viruses have been detected in virtually all cervical cancers. C-33A is one of the rare cervical cancer derived cell lines considered as HPV-negative. Employing monoclonal antibodies raised against a conformational epitope of the HPV-16 E7 oncoprotein, we present evidence suggesting that E7-positive cells can be sporadically and transiently detected in C-33A cell cultures. Immunoblotting with affinity-purified rabbit polyclonal anti-HPV 16 E7 antisera and q-RT-PCR analysis suggest that these cells do probably not express HPV-16 E7. Moreover, we show that the HPV E7 protein level differs considerably between individual cells in cultures of several established cervical cancer cell lines. Our data suggest that expression of the E7 protein is variable in established cervical cancer cell lines including C-33A cells.

Role of C/EBPß-LAP and C/EBPß-LIP in early adipogenic differentiation of human white adipose-derived progenitors and at later stages in immature adipocytes.

We investigated the role of the major isoforms of CCAAT enhancer binding protein ß (C/EBPß), C/EBPß-LAP and C/EBPß-LIP, in adipogenesis of human white adipose-derived stromal/progenitor cells (ASC). C/EBPß gene expression was transiently induced early in adipogenesis. At later stages, in immature adipocytes, the C/EBPß mRNA and protein levels declined. The C/EBPß-LIP protein steady-state level decreased considerably stronger than the C/EBPß-LAP level and the C/EBPß-LIP half-life was significantly shorter than the C/EBPß-LAP half-life. The turn-over of both C/EBPß-isoforms was regulated by ubiquitin/proteasome-dependent degradation. These data suggest that the protein stability of the C/EBPß-isoforms is differentially regulated in the course of adipogenesis and in immature adipocytes. Constitutive overexpression of C/EBPß-LIP had antiadipogenic activity in human ASC. C/EBPß-LAP, which promotes adipogenesis in mouse 3T3-L1 preadipocytes by directly activating expression of the adipogenic keyregulator PPARγ2, induced the expression of PPARγ2 and of the adipocyte differentiation gene product FABP4 in confluent ASC in the absence of adipogenic hormones. At later stages after hormone cocktail-induced adipogenesis, in immature adipocytes, constitutive overexpression of C/EBPß-LAP led to reduced expression of PPARγ2 and FABP4, C/EBPα expression was downregulated and the expression of the adipocyte differentiation gene products adiponectin and leptin was impaired. These findings suggest that constitutive overexpression of C/EBPß-LAP induces adipogenesis in human ASC and negatively regulates the expression of adipogenic regulators and certain adipocyte differentiation gene products in immature adipocytes. We conclude the regulation of both C/EBPß gene expression and C/EBPß-LIP and C/EBPß-LAP protein turn-over plays an important role for the expression of adipogenic regulators and/or adipocyte differentiation genes in early adipogenic differentiation of human ASC and at later stages in human immature adipocytes.

Carbonic anhydrase III regulates peroxisome proliferator-activated receptor-γ2.

Carbonic anhydrase III (CAIII) is an isoenzyme of the CA family. Because of its low specific anhydrase activity, physiological functions in addition to hydrating CO(2) have been proposed. CAIII expression is highly induced in adipogenesis and CAIII is the most abundant protein in adipose tissues. The function of CAIII in both preadipocytes and adipocytes is however unknown. In the present study we demonstrate that adipogenesis is greatly increased in mouse embryonic fibroblasts (MEFs) from CAIII knockout (KO) mice, as demonstrated by a greater than 10-fold increase in the induction of fatty acid-binding protein-4 (FABP4) and increased triglyceride formation in CAIII(-/-) MEFs compared with CAIII(+/+) cells. To address the underlying mechanism, we investigated the expression of the two adipogenic key regulators, peroxisome proliferator-activated receptor-γ2 (PPARγ2) and CCAAT/enhancer binding protein-α. We found a considerable (approximately 1000-fold) increase in the PPARγ2 expression in the CAIII(-/-) MEFs. Furthermore, RNAi-mediated knockdown of endogenous CAIII in NIH 3T3-L1 preadipocytes resulted in a significant increase in the induction of PPARγ2 and FABP4. When both CAIII and PPARγ2 were knocked down, FABP4 was not induced. We conclude that down-regulation of CAIII in preadipocytes enhances adipogenesis and that CAIII is a regulator of adipogenic differentiation which acts at the level of PPARγ2 gene expression.

Analysis of the cellular uptake and nuclear delivery of insulin-like growth factor binding protein-3 in human osteosarcoma cells.

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) regulates cell proliferation and survival by extracellular interaction and inactivation of the growth factor IGF-I. Beyond that, IGF-independent actions mediated by intracellular IGFBP-3 including nuclear-IGFBP-3, have also been described. We here show, using both confocal and electron microscopy and cell fractionation, that the extracellular addition of IGFBP-3 to living cells results in rapid uptake and nuclear delivery of IGFBP-3, by yet partly unknown mechanisms. IGFBP-3 is internalized through a dynamin-dependent pathway, traffics through endocytic compartments and is finally delivered into the nucleus. We observed docking of IGFBP-3 containing structures to the nuclear envelope and found IGFBP-3 containing dot-like structures to permeate the nuclear envelope. In summary, our findings establish the pathway by which this tumor suppressor protein is delivered from extracellular space to the nucleus.

Detection of human papillomavirus type 18 E7 oncoprotein in cervical smears: a feasibility study.

Persistent infections by high-risk human papillomaviruses (HPVs) are the main etiological factor for cervical cancer, and expression of HPV E7 oncoproteins was suggested to be a potential marker for tumor progression. The objective of this study was to generate new reagents for the detection of the HPV18 E7 oncoprotein in cervical smears. Rabbit monoclonal antibodies against recombinant E7 protein of HPV type 18 (HPV18) were generated and characterized using Western blotting, epitope mapping, indirect immunofluorescence, and immunohistochemistry. One clone specifically recognizing HPV18 E7 was used for the development of a sandwich enzyme-linked immunosorbent assay (ELISA). The assay was validated using recombinant E7 proteins of various HPV types and lysates from E7-positive cervical carcinoma cells. A total of 14 HPV18 DNA-positive cervical swab specimens and 24 HPV DNA-negative-control specimens were used for the determination of E7 protein levels by the newly established sandwich ELISA. On the basis of the average absorbance values obtained from all 24 negative controls, a cutoff above which a clinical sample can be judged E7 positive was established. Significant E7 signals 6- to 30-fold over background were found in 7 out of 14 abnormal HPV18 DNA-positive cervical smear specimens. This feasibility study demonstrates for the first time that HPV18 E7 oncoprotein can be detected in cervical smears.

Dipeptidyl peptidase-4 cell surface expression marks an abundant adipose stem/progenitor cell population with high stemness in human white adipose tissue.

The capacity of adipose stem/progenitor cells (ASCs) to undergo self-renewal and differentiation is crucial for adipose tissue homoeostasis, regeneration and expansion. However, the heterogeneous ASC populations of the adipose lineage constituting adipose tissue are not precisely known. In the present study, we demonstrate that cell surface expression of dipeptidyl peptidase-4 (DPP4)/cluster of differentiation 26 (CD26) subdivides the DLK1-/CD34+/CD45-/CD31- ASC pool of human white adipose tissues (WATs) into two large populations. Ex vivo, DPP4+ ASCs possess higher self-renewal and proliferation capacity and lesser adipocyte differentiation potential than DDP4- ASCs. The knock-down of DPP4 in ASC leads to significantly reduced proliferation and self-renewal capacity, while adipogenic differentiation is increased. Ectopic overexpression of DPP4 strongly inhibits adipogenesis. Moreover, in whole mount stainings of human subcutaneous (s)WAT, we detect DPP4 in CD34+ ASC located in the vascular stroma surrounding small blood vessels and in mature adipocytes. We conclude that DPP4 is a functional marker for an abundant ASC population in human WAT with high proliferation and self-renewal potential and low adipogenic differentiation capacity.

An organoid model derived from human adipose stem/progenitor cells to study adipose tissue physiology.

We established a functional adipose organoid model system for human adipose stem/progenitor cells (ASCs) isolated from white adipose tissue (WAT). ASCs were forced to self-aggregate by a hanging-drop technique. Afterwards, spheroids were transferred into agar-coated cell culture dishes to avoid plastic-adherence and dis-aggregation. Adipocyte differentiation was induced by an adipogenic hormone cocktail. Morphometric analysis revealed a significant increase in organoid size in the course of adipogenesis until d 18. Whole mount staining of organoids using specific lipophilic dyes showed large multi- and unilocular fat deposits in differentiated cells indicating highly efficient differentiation of ASCs into mature adipocytes. Moreover, we found a strong induction of the expression of key adipogenesis and adipocyte markers (CCAAT/enhancer-binding protein (C/EBP) ß, peroxisome proliferator-activated receptor (PPAR) γ, fatty acid-binding protein 4 (FABP4), adiponectin) during adipose organoid formation. Secreted adiponectin was detected in the cell culture supernatant, underscoring the physiological relevance of mature adipocytes in the organoid model. Moreover, colony formation assays of collagenase-digested organoids revealed the maintenance of a significant fraction of ASCs within newly formed organoids. In conclusion, we provide a reliable and highly efficient WAT organoid model, which enables accurate analysis of cellular and molecular markers of adipogenic differentiation and adipocyte physiology.

The circadian transcription factor ARNTL2 is regulated by weight-loss interventions in human white adipose tissue and inhibits adipogenesis.

Misalignment of physiological circadian rhythms promotes obesity which is characterized by white adipose tissue (WAT) expansion. Differentiation of Adipose stem/progenitor cells (ASCs) contributes to WAT increase but the importance of the cellular clock in this process is incompletely understood. In the present study, we reveal the role of the circadian transcription factor Aryl hydrocarbon receptor nuclear translocator-like 2 (ARNTL2) in human ASCs, isolated from subcutaneous (s)WAT samples of patients undergoing routine elective plastic abdominal surgery. We show that circadian synchronization by serum-shock or stimulation with adipogenic stimuli leads to a different expression pattern of ARNTL2 relative to its well-studied paralogue ARNTL1. We demonstrate that ARNTL2 mRNA is downregulated in ASCs upon weight-loss (WL) whereas ARNTL2 protein is rapidly induced in the course of adipogenic differentiation and highly abundant in adipocytes. ARNTL2 protein is maintained in ASCs cooperatively by mechanistic Target of Rapamycin (mTOR) and Mitogen-activated Protein Kinase (MAPK) signalling pathways while ARNTL2 functions as an inhibitor on both circuits, leading to a feedback mechanism. Consistently, ectopic overexpression of ARNTL2 repressed adipogenesis by facilitating the degradation of ARNTL1, inhibition of Kruppel-Like Factor 15 (KLF15) gene expression and down-regulation of the MAPK-CCAAT/enhancer-binding protein ß (C/EBPß) axis. Western blot analysis of sWAT samples from normal-weight, obese and WL donors revealed that ARNTL2 protein was solely elevated by WL compared to ARNTL1 which underscores unique functions of both transcription factors. In conclusion, our study reveals ARNTL2 to be a WL-regulated inhibitor of adipogenesis which might provide opportunities to develop strategies to ameliorate obesity.

Targeting cellular senescence based on interorganelle communication, multilevel proteostasis, and metabolic control.

Cellular senescence, a stable cell division arrest caused by severe damage and stress, is a hallmark of aging in vertebrates including humans. With progressing age, senescent cells accumulate in a variety of mammalian tissues, where they contribute to tissue aging, identifying cellular senescence as a major target to delay or prevent aging. There is an increasing demand for the discovery of new classes of small molecules that would either avoid or postpone cellular senescence by selectively eliminating senescent cells from the body (i.e., 'senolytics') or inactivating/switching damage-inducing properties of senescent cells (i.e., 'senostatics/senomorphics'), such as the senescence-associated secretory phenotype. Whereas compounds with senolytic or senostatic activity have already been described, their efficacy and specificity has not been fully established for clinical use yet. Here, we review mechanisms of senescence that are related to mitochondria and their interorganelle communication, and the involvement of proteostasis networks and metabolic control in the senescent phenotype. These cellular functions are associated with cellular senescence in in vitro and in vivo models but have not been fully exploited for the search of new compounds to counteract senescence yet. Therefore, we explore possibilities to target these mechanisms as new opportunities to selectively eliminate and/or disable senescent cells with the aim of tissue rejuvenation. We assume that this research will provide new compounds from the chemical space which act as mimetics of caloric restriction, modulators of calcium signaling and mitochondrial physiology, or as proteostasis optimizers, bearing the potential to counteract cellular senescence, thereby allowing healthy aging.

Quiescence, Stemness and Adipogenic Differentiation Capacity in Human DLK1-/CD34+/CD24+ Adipose Stem/Progenitor Cells.

We explore the status of quiescence, stemness and adipogenic differentiation capacity in adipose stem/progenitor cells (ASCs) ex vivo, immediately after isolation from human subcutaneous white adipose tissue, by sorting the stromal vascular fraction into cell-surface DLK1+/CD34-, DLK1+/CD34dim and DLK1-/CD34+ cells. We demonstrate that DLK1-/CD34+ cells, the only population exhibiting proliferative and adipogenic capacity, express ex vivo the bonafide quiescence markers p21Cip1, p27Kip1 and p57Kip2 but neither proliferation markers nor the senescence marker p16Ink4a. The pluripotency markers NANOG, SOX2 and OCT4 are barely detectable in ex vivo ASCs while the somatic stemness factors, c-MYC and KLF4 and the early adipogenic factor C/EBPß are highly expressed. Further sorting of ASCs into DLK1-/CD34+/CD24- and DLK1-/CD34+/CD24+ fractions shows that KLF4 and c-MYC are higher expressed in DLK1-/CD34+/CD24+ cells correlating with higher colony formation capacity and considerably lower adipogenic activity. Proliferation capacity is similar in both populations. Next, we show that ASCs routinely isolated by plastic-adherence are DLK1-/CD34+/CD24+. Intriguingly, CD24 knock-down in these cells reduces proliferation and adipogenesis. In conclusion, DLK1-/CD34+ ASCs in human sWAT exist in a quiescent state, express high levels of somatic stemness factors and the early adipogenic transcription factor C/EBPß but senescence and pluripotency markers are barely detectable. Moreover, our data indicate that CD24 is necessary for adequate ASC proliferation and adipogenesis and that stemness is higher and adipogenic capacity lower in DLK1-/CD34+/CD24+ relative to DLK1-/CD34+/CD24- subpopulations.

A new method for the purification of bioactive insulin-like growth factor-binding protein-3.

We present a novel efficient procedure for high level purification of human IGFBP-3. Insulin-like growth factor-binding proteins (IGFBPs) are key regulators of insulin-like growth factor mediated signal transduction and thereby can profoundly influence cellular phenotypes. Certain IGFBPs, including IGFBP-3, have also been described to possess additional IGF-independent activities, which rely, at least in part, on their nuclear localization. However, the mechanisms of IGF-independent biological activities of IGFBP-3 are not well understood. For the study of these functions, recombinant IGFBP-3 is used. However, it has traditionally been difficult to obtain recombinant protein in sufficient quality and quantity. Here we describe a new procedure for the purification of recombinant IGFBP-3 from cell culture supernatants involving a two-step affinity purification procedure. Using this new protocol, we obtained pure IGFBP-3 free of any visible contaminants. We also provide evidence that the protein purified in this way retains biological activity, to bind IGF and modulate IGF-dependent signal transduction. We also show that the purified protein produced by the new procedure is readily internalized by human fibroblasts, suggesting that this protein is also suitable to study intracellular trafficking of IGFBP-3.

Pyruvate kinase isoenzyme M2 is a glycolytic sensor differentially regulating cell proliferation, cell size and apoptotic cell death dependent on glucose supply.

The glycolytic key regulator pyruvate kinase M2 (M2-PK or PKM2) can switch between a highly active tetrameric and an inactive dimeric form. The transition between the two conformations regulates the glycolytic flux in tumor cells. We developed specific M2-PK-binding peptide aptamers which inhibit M2-PK, but not the 96% homologous M1-PK isoenzyme. In this study we demonstrate that, at normal blood glucose concentrations, peptide aptamer-mediated inhibition of M2-PK induces a significant decrease of the population doubling (PDL rate) and cell proliferation rate as well as an increase in cell size, whereas under glucose restriction an increase in PDL and cell proliferation rates but a decrease in cell size was observed. Moreover, M2-PK inhibition rescues cells from glucose starvation-induced apoptotic cell death by increasing the metabolic activity. These findings suggest that M2-PK is a metabolic sensor which regulates cell proliferation, cell growth and apoptotic cell death in a glucose supply-dependent manner.

Sprouty1 Prevents Cellular Senescence Maintaining Proliferation and Differentiation Capacity of Human Adipose Stem/Progenitor Cells.

The role of Ras-Mitogen-activated protein kinase (MAPK) signaling in cellular aging is not precisely understood. Recently, we identified Sprouty1 (SPRY1) as a weight-loss target gene in human adipose stem/progenitor cells (ASCs) and showed that Sprouty1 is important for proper regulation of adipogenesis. In the present study, we show that loss-of-function of Sprouty1 by CRISPR/Cas9-mediated genome editing in human ASCs leads to hyper-activation of MAPK signaling and a senescence phenotype. Sprouty1 knockout ASCs undergo an irreversible cell cycle arrest, become enlarged and stain positive for senescence-associated ß-galactosidase. Sprouty1 down-regulation leads to DNA double strand breaks, a considerably increased number of senescence-associated heterochromatin foci and induction of p53 and p21Cip1. In addition, we detect an increase of hypo-phosphorylated Retinoblastoma (Rb) protein in SPRY1 knockout ASCs. p16Ink4A is not induced. Moreover, we show that Sprouty1 knockout leads to induction of a senescence-associated secretory phenotype as indicated by the activation of the transcription factors NFκB and C/EBPß and a significant increase in mRNA expression and secretion of interleukin-8 (IL-8) and CXCL1/GROα. Finally, we demonstrate that adipogenesis is abrogated in senescent SPRY1 knockout ASCs. In conclusion, this study reveals a novel mechanism showing the importance of Sprouty1 for the prevention of senescence and the maintenance of the proliferation and differentiation capacity of human ASCs.

CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.

The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 (SPRY1) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/Cas9 target sequences and the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the SPRY1 gene is determined by a PCR-based mutation detection assay and sequence analysis. Effects on mRNA and protein levels are studied by RT-qPCR and Western blotting. In addition, we demonstrate that CRISPR/Cas9 mediated SPRY1 KO and gene silencing by shRNA are similarly effective to deplete the Sprouty1 protein and to inhibit adipogenic differentiation. In summary, we show a reliable approach to achieve a gene KO in human ASCs, which could also apply to other primary cell types. Abbreviations: ASC: Adipogenic Stem/Progenitor Cell; Cas: CRISPR-associated system; CRISPR: Clustered Regularly Interspaced Palindromic Repeat; gDNA: Genomic DNA; GOI: Gene of interest; gRNA: Guide RNA; NHEJ: Non-homologous end joining; Indel: Insertion/Deletion; PAM: Protospacer adjacent motif; sWAT: Subcutaneous white adipose tissue; TIDE: Tracking of indels by decomposition.

The SUMO-E3 ligase PIAS3 targets pyruvate kinase M2.

Pyruvate kinase M2 (M2-PK) controls the rate-limiting step at the end of the glycolytic pathway in normal proliferating and tumor cells. Other functions of M2-PK in addition to its role in glycolysis are little understood. The aim of this study was to identify new cellular interaction partners of M2-PK in order to discover novel links between M2-PK and cellular functions. Here we show that the SUMO-E3 ligase protein PIAS3 (inhibitor of activated STAT3) physically interacts with M2-PK and its isoenzyme M1-PK. Moreover, we demonstrate that endogenous SUMO-1-M2-PK conjugates exist in mammalian cells. Furthermore, we show that transient expression of PIAS3 but not the RING domain mutant PIAS3 (C299S, H301A) is consistent with nuclear localization of M2-PK and PIAS3 and M2-PK partially co-localize in the nucleus of these cells. This study suggests a link between PIAS3 and nuclear pyruvate kinase.