scholarly journals Only the large soluble form of preadipocyte factor-1 (Pref-1), but not the small soluble and membrane forms, inhibits adipocyte differentiation: role of alternative splicing

2002 ◽  
Vol 364 (1) ◽  
pp. 137-144 ◽  
Author(s):  
Baisong MEI ◽  
Ling ZHAO ◽  
Li CHEN ◽  
Hei Sook SUL
Keyword(s):  
Alternative Splicing ◽  
Adipocyte Differentiation ◽  
Constitutive Expression ◽  
Conditioned Media ◽  
Full Length ◽  
Biologically Active ◽  
Soluble Form ◽  
Cos Cells ◽  
Splicing Variants ◽  
Processing Site

We originally identified preadipocyte factor-1 (Pref-1) as an inhibitor of adipogenesis by the fact that constitutive expression of full-length Pref-1A inhibits differentiation of 3T3-L1 cells into adipocytes. Subsequently, we found that the membrane form of Pref-1 is proteolytically processed at two sites in the extracellular domain, resulting in the larger (50kDa) and smaller (25kDa) soluble forms. A specific form(s) of Pref-1, which is active in inhibiting adipocyte differentiation, has not been elucidated. Here, various artificial constructs and alternative-splicing variants of Pref-1 were stably transfected into 3T3-L1 cells, or conditioned media from COS cells transfected with the various forms were added into differentiating 3T3-L1 cells. Judging by Oil Red O staining for lipid accumulation and expression of adipocyte markers, we determined that, unlike the full-length Pref-1A and the constructed large soluble form, the artificial membrane form of Pref-1 lacking the processing site proximal to the membrane was not effective in inhibiting adipogenesis. Furthermore, conditioned media from COS cells transfected with the construct containing only the first three epidermal growth factor repeats, corresponding to the small soluble form, was not effective in inhibiting adipocyte differentiation. Of the four alternative-splicing products, Pref-1A and Pref-1B, which generate both large and small soluble forms, inhibited adipogenesis, whereas Pref-1C and Pref-1D, which lack the processing site proximal to the membrane and therefore generate only the smaller soluble form, did not show any effect. We conclude that only the large soluble form, and not the transmembrane or the small soluble form, of Pref-1 is biologically active and that alternative splicing therefore determines Pref-1 function in adipocyte differentiation.

scholarly journals Cleavage of membrane-associated pref-1 generates a soluble inhibitor of adipocyte differentiation.

1997 ◽  
Vol 17 (2) ◽  
pp. 977-988 ◽  
Author(s):  
C M Smas ◽  
L Chen ◽  
H S Sul
Keyword(s):  
Adipocyte Differentiation ◽  
Constitutive Expression ◽  
Soluble Form ◽  
Inhibitory Effects ◽  
Alternate Splicing ◽  
Produce Cell ◽  
Present Evidence ◽  
Juxtamembrane Region ◽  
Epidermal Growth ◽  
First Time

pref-1 is an epidermal growth factor-like repeat protein present on the surface of preadipocytes that functions in the maintenance of the preadipose state. pref-1 expression is completely abolished during 3T3-L1 adipocyte differentiation. Bypassing this downregulation by constitutive expression of full-length transmembrane pref-1 in preadipocytes drastically inhibits differentiation. For the first time, we show processing of cell-associated pref-1 to generate both a soluble pref-1 protein of approximately 50 kDa that corresponds to the ectodomain and also smaller products of 24 to 25 kDa and 31 kDa. Furthermore, while all four of the alternately spliced forms of pref-1 produce cell-associated protein, only the two largest of the four alternately spliced isoforms undergo cleavage in the juxtamembrane region to release the soluble 50-kDa ectodomain. We demonstrate that addition of Escherichia coli-expressed pref-1 ectodomain to 3T3-L1 preadipocytes blocks differentiation, thus overriding the adipogenic actions of dexamethasone and methylisobutylxanthine. The inhibitory effects of the pref-1 ectodomain are blocked by preincubation of the protein with pref-1 antibody. That the ectodomain alone is sufficient for inhibition demonstrates that transmembrane pref-1 can be processed to generate an inhibitory soluble form, thereby greatly extending its range of action. Furthermore, we present evidence that alternate splicing is the mechanism that governs the production of transmembrane versus soluble pref-1, thereby determining the mode of action, juxtacrine or paracrine, of the pref-1 protein.

scholarly journals Pref-1 Interacts with Fibronectin To Inhibit Adipocyte Differentiation

2010 ◽  
Vol 30 (14) ◽  
pp. 3480-3492 ◽  
Author(s):  
Yuhui Wang ◽  
Ling Zhao ◽  
Cynthia Smas ◽  
Hei Sook Sul
Keyword(s):  
Adipocyte Differentiation ◽  
Transmembrane Protein ◽  
Dominant Negative ◽  
Biologically Active ◽  
Mitogen Activated Protein Kinase ◽  
Soluble Form ◽  
Necrosis Factor Alpha ◽  
Rgd Peptides ◽  
Downstream Signaling ◽  
Erk Mapk

ABSTRACT Pref-1/Dlk1 is made as an epidermal growth factor (EGF) repeat-containing transmembrane protein but is cleaved by tumor necrosis factor alpha converting enzyme (TACE) to generate a biologically active soluble form. Soluble Pref-1 inhibits adipocyte differentiation through the activation of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and the subsequent upregulation of Sox9 expression. However, others have implicated Notch in Pref-1 signaling and function. Here, we show that Pref-1 does not interact with, or require, Notch for its function. Instead, we show a direct interaction of Pref-1 and fibronectin via the Pref-1 juxtamembrane domain and fibronectin C-terminal domain. We also show that fibronectin is required for the Pref-1-mediated inhibition of adipocyte differentiation, the activation of ERK/MAPK, and the upregulation of Sox9. Furthermore, disrupting fibronectin binding to integrin by the addition of RGD peptides or by the knockdown of α5 integrin prevents the Pref-1 inhibition of adipocyte differentiation. Pref-1 activates the integrin downstream signaling molecules, FAK and Rac, and ERK activation by Pref-1 is blunted by the knockdown of Rac or by the forced expression of dominant-negative Rac. We conclude that, by interacting with fibronectin, Pref-1 activates integrin downstream signaling to activate MEK/ERK and to inhibit adipocyte differentiation.

scholarly journals Pref-1 (Preadipocyte Factor 1) Activates the MEK/Extracellular Signal-Regulated Kinase Pathway To Inhibit Adipocyte Differentiation

2006 ◽  
Vol 27 (6) ◽  
pp. 2294-2308 ◽  
Author(s):  
Kyung-Ah Kim ◽  
Jung-Hyun Kim ◽  
Yuhui Wang ◽  
Hei Sook Sul
Keyword(s):  
Adipocyte Differentiation ◽  
Transmembrane Protein ◽  
Biologically Active ◽  
Soluble Form ◽  
Null Mouse ◽  
Dependent Manner ◽  
Wild Type ◽  
Extracellular Signal Regulated Kinase ◽  
Erk Phosphorylation ◽  
Extracellular Signal

ABSTRACT Preadipocyte factor 1 (Pref-1) is found in preadipocytes but is absent in adipocytes. Pref-1 is made as a transmembrane protein but is cleaved to generate a biologically active soluble form. Although Pref-1 inhibition of adipogenesis has been well studied in vitro and in vivo, the signaling pathway for Pref-1 is not known. Here, by using purified soluble Pref-1 in Pref-1 null mouse embryo fibroblasts (MEF), we show that Pref-1 increases MEK/extracellular signal-regulated kinase (ERK) phosphorylation in a time- and dose-dependent manner. Compared to wild-type MEF, differentiation of Pref-1 null MEF into adipocytes is enhanced, as judged by lipid accumulation and adipocyte marker expression. Both wild-type and Pref-1 null MEF show a transient burst of ERK phosphorylation upon addition of adipogenic agents. Wild-type MEF show a significant, albeit lower, second increase in ERK phosphorylation peaking at day 2. This ERK phosphorylation, corresponding to Pref-1 abundance, is absent during differentiation of Pref-1 null MEF. Prevention of this second increase in ERK1/2 phosphorylation in wild-type MEF by the MEK inhibitor PD98059 or by transient depletion of ERK1/2 via small interfering RNA-enhanced adipocyte differentiation. Furthermore, treatment of Pref-1 null MEF with Pref-1 restores this ERK phosphorylation, resulting in inhibition of adipocyte differentiation primarily by preventing peroxisome proliferator-activated receptor γ2 induction. However, in the presence of PD98059 or depletion of ERK1/2, exogenous Pref-1 cannot inhibit adipocyte differentiation in Pref-1 null MEF. We conclude that Pref-1 activates MEK/ERK signaling, which is required for Pref-1 inhibition of adipogenesis.

Characterisation of novel splicing variants of the tyrosine hydroxylase C-terminal domain in human neuroblastic tumours

2007 ◽  
Vol 388 (4) ◽  
pp. 419-426 ◽  
Author(s):  
Josep Roma ◽  
Ester Saus ◽  
Marc Cuadros ◽  
Jaume Reventós ◽  
Josep Sánchez de Toledo ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Tyrosine Hydroxylase ◽  
Adrenal Glands ◽  
Full Length ◽  
General Distribution ◽  
Western Blots ◽  
Terminal Domain ◽  
Splicing Variants ◽  
First Time ◽  
Novel Isoforms

Abstract Alternative splicing of human tyrosine hydroxylase (hTH) transcripts appears to occur mainly in the N-terminal domain, giving rise to at least eight different isoforms. We recently reported the existence of hTH transcript variants resulting from splicing of exons 8 and 9, within a region previously thought to be constant. The mRNA distribution of these novel hTH isoforms in neuroblastic tumours and in foetal adrenal glands was analysed by conventional and real-time RT-PCR. The presence of the target protein was determined by Western blotting, immunoprecipitation and protein analysis. Transcripts lacking exons 8 and 9 were widely distributed in the tissues analysed. Characterisation of full-length mRNA revealed that splicing of exons 8 and 9 was always associated with splicing of exons 2 (hTH-Δ2,8,9) or 1b and 2 (hTH-Δ1b,2,8,9). In addition, one variant detected on Western blots in several tumours fits the predicted size (58 kDa) of the isoforms lacking exons 8 and 9. In conclusion, the two novel isoforms reported here (hTH-Δ2,8,9 and hTH-Δ1b,2,8,9) represent the first full-length isoforms with alternative splicing in the hTH C-terminal domain. These results demonstrate for the first time the existence of hTH isoforms Δ2,8,9 and Δ1b,2,8,9. Their general distribution in neuroblastoma and adrenal glands and translation into protein suggest a significant functional role for these novel hTH isoforms, which merit further study.

scholarly journals Dual regulation of telomerase activity through c-Myc-dependent inhibition and alternative splicing of hTERT

2002 ◽  
Vol 115 (6) ◽  
pp. 1305-1312 ◽  
Author(s):  
Ana Cerezo ◽  
Holger Kalthoff ◽  
Markus Schuermann ◽  
Birgit Schäfer ◽  
Petra Boukamp
Keyword(s):  
Alternative Splicing ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Constitutive Expression ◽  
Telomerase Activity ◽  
Full Length ◽  
General Mechanism ◽  
Htert Expression ◽  
Dominant Form ◽  
Tgf Β1

Telomerase is believed to be induced upon proliferation and inhibited when cells differentiate. Thus, regulation of telomerase activity could be an important mechanism to limit growth of normal and cancer cells. Using transforming growth factor-beta 1 (TGF-β1), which is known to control proliferation in epithelial cells, we now demonstrate that in the human HaCaT skin keratinocytes, TGF-β1 downregulated c-Myc, and this blocked proliferation. This also caused a decrease in hTERT expression, which in turn inhibited telomerase activity. Overexpressing hTERT recovered telomerase activity but not proliferation, whereas constitutive expression of c-Myc recovered proliferation and hTERT expression. Nevertheless, telomerase remained inhibited, thus dissociating proliferation and telomerase activity. In addition, we show that TGF-β1 inhibited telomerase activity despite ongoing hTERT transcription by inducing loss of the full-length hTERT transcript (mediating telomerase activity) and retaining high expression of the inactive β variant. These changes in the splicing pattern reversed upon TGF-β1 removal, as did inhibition of telomerase activity, suggesting that alternative splicing may represent a novel mechanism of telomerase regulation by TGF-β1. In addition, we show that destruction of tissue integrity (in a model for epidermal blistering) resulted in a rapid induction of the inactive β variant, whereas tissue regeneration (formation of a stratified epithelium) correlated with a shift to the active full-length transcript, which is the dominant form in intact epidermis. Thus alternative splicing may not be restricted to TGF-β1 but may add a more general mechanism of hTERT regulation in epidermal cells.

scholarly journals Alternative splicing of interleukin-33 and type 2 inflammation in asthma

2016 ◽  
Vol 113 (31) ◽  
pp. 8765-8770 ◽  
Author(s):  
Erin D. Gordon ◽  
Laura J. Simpson ◽  
Cydney L. Rios ◽  
Lando Ringel ◽  
Marrah E. Lachowicz-Scroggins ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Mast Cells ◽  
Airway Epithelial Cells ◽  
Full Length ◽  
Biologically Active ◽  
Target Cells ◽  
Interleukin 33 ◽  
Type 2 Inflammation

Type 2 inflammation occurs in a large subgroup of asthmatics, and novel cytokine-directed therapies are being developed to treat this population. In mouse models, interleukin-33 (IL-33) activates lung resident innate lymphoid type 2 cells (ILC2s) to initiate airway type 2 inflammation. In human asthma, which is chronic and difficult to model, the role of IL-33 and the target cells responsible for persistent type 2 inflammation remain undefined. Full-length IL-33 is a nuclear protein and may function as an “alarmin” during cell death, a process that is uncommon in chronic stable asthma. We demonstrate a previously unidentified mechanism of IL-33 activity that involves alternative transcript splicing, which may operate in stable asthma. In human airway epithelial cells, alternative splicing of the IL-33 transcript is consistently present, and the deletion of exons 3 and 4 (Δ exon 3,4) confers cytoplasmic localization and facilitates extracellular secretion, while retaining signaling capacity. In nonexacerbating asthmatics, the expression of Δ exon 3,4 is strongly associated with airway type 2 inflammation, whereas full-length IL-33 is not. To further define the extracellular role of IL-33 in stable asthma, we sought to determine the cellular targets of its activity. Comprehensive flow cytometry and RNA sequencing of sputum cells suggest basophils and mast cells, not ILC2s, are the cellular sources of type 2 cytokines in chronic asthma. We conclude that IL-33 isoforms activate basophils and mast cells to drive type 2 inflammation in chronic stable asthma, and novel IL-33 inhibitors will need to block all biologically active isoforms.

Full-length-transcriptomic analysis in mice and human heart using Single-Molecule Real-time Sequencing (SMRT) identified 15 novel isoforms and a novel promoter region of PGC1-alpha

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
D Oehler ◽  
A Goedecke ◽  
A Spychala ◽  
K Lu ◽  
N Gerdes ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Single Molecule ◽  
High Fat Diet ◽  
Human Heart ◽  
Full Length ◽  
Funding Source ◽  
Smrt Sequencing ◽  
High Fat ◽  
Exon 1 ◽  
Novel Exon

Abstract Background Alternative splicing is a process by which exons within a pre-mRNA are joined or skipped, resulting in isoforms being encoded by a single gene. Alternative Splicing affecting transcription factors may have substantial impact on cellular dynamics. The PPARG Coactivator 1 Alpha (PGC1-α), is a major modulator in energy metabolism. Data from murine skeletal muscle revealed distinctive isoform patterns giving rise to different phenotypes, i.e. mitogenesis and hypertrophy. Here, we aimed to establish a complete dataset of isoforms in murine and human heart applying single-molecule real-time (SMRT)-sequencing as novel approach to identify transcripts without need for assembly, resulting in true full-length sequences. Moreover, we aimed to unravel functional relevance of the various isoforms during experimental ischemia reperfusion (I/R). Methods RNA-Isolation was performed in murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery), followed by library preparation and SMRT-Sequencing. Bioinformatic analysis was done using a modified IsoSeq3-Pipeline and OS-tools. Identification of PGC1-α isoforms was fulfilled by similarity search against exonic sequences within the full-length, non-concatemere (FLNC) reads. Isoforms with Open-Reading-Frame (ORF) were manually curated and validated by PCR and Sanger-Sequencing. I/R was induced by ligature of the LAD for 45 min in mice on standard chow as well as on high-fat-high-sucrose diet. Area At Risk (AAR) and remote tissue were collected three and 16 days after I/R or sham-surgery (n=4 per time point). Promotor patterns were analyzed by qPCR. Results Deciphering the full-length transcriptome of murine and human heart resulted in ∼60000 Isoforms with 99% accuracy on mRNA-sequence. Focusing on murine PGC1-α-isoforms we discovered and verified 15 novel transcripts generated by hitherto unknown splicing events. Additionally, we identified a novel Exon 1 originating between the known promoters followed by a valid ORF, suggesting the discovery of a novel promoter. Remarkably, we found a homologous novel Exon1 in human heart, suggesting conservation of the postulated promoter. In I/R the AAR exhibited a significant lower expression of established and novel promoters compared to remote under standard chow 3d post I/R. 16d post I/R, the difference between AAR & Remote equalized in standard chow while remaining under High-Fat-Diet. Conclusion Applying SMRT-technique, we generated the first time a complete full-length-transcriptome of the murine and human heart, identifying 15 novel potentially coding transcripts of PGC1-α and a novel exon 1. These transcripts are differentially regulated in experimental I/R in AAR and remote myocardium, suggesting transcriptional regulation and alternative splicing modulating PGC1-α function in heart. Differences between standard chow and high fat diet suggest impact of impaired glucose metabolism on regulatory processes after myocardial infarction. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Collaborative Research Centre 1116 (German Research Foundation)

scholarly journals Electrospun fibers enhanced the paracrine signaling of mesenchymal stem cells for cartilage regeneration

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nurul Dinah Kadir ◽  
Zheng Yang ◽  
Afizah Hassan ◽  
Vinitha Denslin ◽  
Eng Hin Lee
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Electrospun Fiber ◽  
Cartilage Regeneration ◽  
Conditioned Media ◽  
Biologically Active ◽  
Paracrine Signaling ◽  
Therapeutic Value ◽  
A Cell ◽  
Fiber Sheet

Abstract Background Secretome profiles of mesenchymal stem cells (MSCs) are reflective of their local microenvironments. These biologically active factors exert an impact on the surrounding cells, eliciting regenerative responses that create an opportunity for exploiting MSCs towards a cell-free therapy for cartilage regeneration. The conventional method of culturing MSCs on a tissue culture plate (TCP) does not provide the physiological microenvironment for optimum secretome production. In this study, we explored the potential of electrospun fiber sheets with specific orientation in influencing the MSC secretome production and its therapeutic value in repairing cartilage. Methods Conditioned media (CM) were generated from MSCs cultured either on TCP or electrospun fiber sheets of distinct aligned or random fiber orientation. The paracrine potential of CM in affecting chondrogenic differentiation, migration, proliferation, inflammatory modulation, and survival of MSCs and chondrocytes was assessed. The involvement of FAK and ERK mechanotransduction pathways in modulating MSC secretome were also investigated. Results We showed that conditioned media of MSCs cultured on electrospun fiber sheets compared to that generated from TCP have improved secretome yield and profile, which enhanced the migration and proliferation of MSCs and chondrocytes, promoted MSC chondrogenesis, mitigated inflammation in both MSCs and chondrocytes, as well as protected chondrocytes from apoptosis. Amongst the fiber sheet-generated CM, aligned fiber-generated CM (ACM) was better at promoting cell proliferation and augmenting MSC chondrogenesis, while randomly oriented fiber-generated CM (RCM) was more efficient in mitigating the inflammation assault. FAK and ERK signalings were shown to participate in the modulation of MSC morphology and its secretome production. Conclusions This study demonstrates topographical-dependent MSC paracrine activities and the potential of employing electrospun fiber sheets to improve the MSC secretome for cartilage regeneration.

scholarly journals Expression of a Truncated Yeast Ccc1 Vacuolar Transporter Increases the Accumulation of Endogenous Iron

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1120
Author(s):  
Raquel Sorribes-Dauden ◽  
María Teresa Martínez-Pastor ◽  
Sergi Puig
Keyword(s):  
Iron Homeostasis ◽  
Sufficient Conditions ◽  
Constitutive Expression ◽  
Iron Bioavailability ◽  
Full Length ◽  
Yeast Cells ◽  
Deficiency Anemia ◽  
Functional Protein ◽  
Amino Terminal ◽  
In Cells

Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in multiple metabolic processes. Iron bioavailability is highly restricted due to the low solubility of its oxidized form, frequently leading to iron deficiency anemia. The baker’s yeast Saccharomyces cerevisiae is used as a model organism for iron homeostasis studies, but also as a food supplement and fermentative microorganism in the food industry. Yeast cells use the vacuolar Ccc1 transporter to detoxify and store excess iron in the vacuoles. Here, we modulate CCC1 expression and properties to increase iron extraction from the environment. We show that constitutive expression of full-length CCC1 is toxic, whereas deletion of its cytosolic amino-terminal (Nt) domain (NtDCCC1) rescues this phenotype. Toxicity is exacerbated in cells lacking AFT1 transcription factor. Further characterization of NtDCcc1 protein suggests that it is a partially functional protein. Western blot analyses indicate that deletion of Ccc1 Nt domain does not significantly alter GFP-Ccc1 protein stability. A functional full-length GFP-Ccc1 protein localized to particular regions of the vacuolar membrane, whereas GFP-NtDCcc1 protein was evenly distributed throughout this endogenous membrane. Interestingly, expression of NtDCCC1 increased the accumulation of endogenous iron in cells cultivated under iron-sufficient conditions, a strategy that could be used to extract iron from media that are not rich in iron.

Sign in / Sign up

Export Citation Format

Share Document