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

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.

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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

Biochemical Journal ◽

10.1042/bj3640137 ◽

2002 ◽

Vol 364 (1) ◽

pp. 137-144 ◽

Cited By ~ 93

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.

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Inhibitory Effects of Fucoidan in 3T3-L1 Adipocyte Differentiation

Marine Biotechnology ◽

10.1007/s10126-008-9170-1 ◽

2008 ◽

Vol 11 (5) ◽

pp. 557-562 ◽

Cited By ~ 57

Author(s):

Mi-Ja Kim ◽

Un-Jae Chang ◽

Jin-Sil Lee

Keyword(s):

Adipocyte Differentiation ◽

Inhibitory Effects

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A Synergistic Anti-Diabetic Effect by Ginsenosides Rb1 and Rg3 through Adipogenic and Insulin Signaling Pathways in 3T3-L1 Cells

Applied Sciences ◽

10.3390/app11041725 ◽

2021 ◽

Vol 11 (4) ◽

pp. 1725

Author(s):

Hee-Do Hong ◽

Sun-Il Choi ◽

Ok-Hwan Lee ◽

Young-Cheul Kim

Keyword(s):

Transcription Factors ◽

Insulin Signaling ◽

Adipocyte Differentiation ◽

Red Ginseng ◽

Inhibitory Effects ◽

Rt Pcr ◽

High Concentration ◽

Expression Of Genes ◽

The Individual ◽

Maximal Expression

Although ginsenosides Rb1 and Rg3 have been identified as the significant ginsenosides found in red ginseng that confer anti-diabetic actions, it is unclear whether insulin-sensitizing effects are mediated by the individual compounds or by their combination. To determine the effect of ginsenosides Rb1 and Rg3 on adipocyte differentiation, 3T3-L1 preadipocytes were induced to differentiate the standard hormonal inducers in the absence or presence of ginsenosides Rb1 or Rg3. Additionally, we determined the effects of Rb1, Rg3, or their combination on the expression of genes related to adipocyte differentiation, adipogenic transcription factors, and the insulin signaling pathway in 3T3-L1 cells using semi-quantitative RT-PCR. Rb1 significantly increased the expression of CEBPα, PPARγ, and aP2 mRNAs. However, Rg3 exerted its maximal stimulatory effect on these genes at 1 μM concentration, while a high concentration (50 μM) showed inhibitory effects. Similarly, treatment with Rb1 and Rg3 (1 μM) increased the expression of IRS-1, Akt, PI3K, GLUT4, and adiponectin. Importantly, co-treatment of Rb1 and Rg3 (9:1) induced the maximal expression levels of these mRNAs. Our data indicate that the anti-diabetic activity of red ginseng is, in part, mediated by synergistic actions of Rb1 and Rg3, further supporting the significance of minor Rg3.

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A novel role for WAVE1 in controlling actin network growth rate and architecture

Molecular Biology of the Cell ◽

10.1091/mbc.e14-10-1477 ◽

2015 ◽

Vol 26 (3) ◽

pp. 495-505 ◽

Cited By ~ 9

Author(s):

Meredith O. Sweeney ◽

Agnieszka Collins ◽

Shae B. Padrick ◽

Bruce L. Goode

Keyword(s):

Actin Filament ◽

Specific Activity ◽

Inhibitory Effect ◽

Actin Network ◽

Inhibitory Effects ◽

Cellular Functions ◽

Network Growth ◽

Assembly Kinetics ◽

Filament Networks ◽

First Time

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 (“V”) domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2.

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Growth factor regulation of uncoupling protein-1 mRNA expression in brown adipocytes

AJP Cell Physiology ◽

10.1152/ajpcell.01396.2000 ◽

2002 ◽

Vol 282 (1) ◽

pp. C105-C112 ◽

Cited By ~ 22

Author(s):

Bibian García ◽

Maria-Jesús Obregón

Keyword(s):

Growth Factor ◽

Adipocyte Differentiation ◽

Uncoupling Protein ◽

Brown Adipocyte ◽

Mrna Levels ◽

Uncoupling Protein 1 ◽

Brown Adipocytes ◽

Proliferation And Differentiation ◽

Epidermal Growth ◽

Continuous Presence

To study the effect of the mitogens epidermal growth factor (EGF), acidic and basic fibroblast growth factors (aFGF and bFGF), and vasopressin on brown adipocyte differentiation, we analyzed the expression of uncoupling protein-1 (UCP-1) mRNA. Quiescent brown preadipocytes express high levels of UCP-1 mRNA in response to triiodothyronine (T3) and norepinephrine (NE). The addition of serum or the mitogenic condition aFGF + vasopressin + NE or EGF + vasopressin + NE decreases UCP-1 mRNA. A second addition of mitogens further decreases UCP-1 mRNA. Treatment with aFGF or bFGF alone increases UCP-1 mRNA, whereas the addition of EGF or vasopressin dramatically reduces UCP-1 mRNA levels. The continuous presence of T3 increases UCP-1 mRNA levels in cells treated with EGF, aFGF, or bFGF. The effect of T3 on the stimulation of DNA synthesis also was tested. T3 inhibits the mitogenic activity of aFGF and bFGF. In conclusion, mitogens like aFGF or bFGF allow brown adipocyte differentiation, whereas EGF and vasopressin inhibit the differentiation process. T3 behaves as an important hormone that regulates both brown adipocyte proliferation and differentiation.

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Regulation of 25-hydroxyvitamin D3-1α-hydroxylase and production of 1α,25-dihydroxyvitamin D3 by human dendritic cells

Blood ◽

10.1182/blood-2002-11-3521 ◽

2003 ◽

Vol 102 (9) ◽

pp. 3314-3316 ◽

Cited By ~ 161

Author(s):

Jana Fritsche ◽

Krishna Mondal ◽

Achim Ehrnsperger ◽

Reinhard Andreesen ◽

Marina Kreutz

Keyword(s):

Dendritic Cells ◽

Early Phase ◽

Constitutive Expression ◽

Terminal Differentiation ◽

Dihydroxyvitamin D3 ◽

Key Enzyme ◽

Functional Consequences ◽

Constitutive Production ◽

Human Dendritic Cells ◽

First Time

Abstract25-Hydroxyvitamin D3-1α-hydroxylase (25(OH)D3-1α-hydroxylase), the key enzyme of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) production, is expressed in monocyte-derived macrophages (MACs). Here we show for the first time constitutive expression of 25(OH)D3-1α-hydroxylase in monocyte-derived dendritic cells (DCs), which was increased after stimulation with lipopolysaccharide (LPS). Accordingly, DCs showed low constitutive production of 1,25(OH)2D3, but activation by LPS increased 1,25(OH)2D3 synthesis. In addition, 25(OH)D3-1α-hydroxylase expression was found in blood DCs but not in CD34+-derived DCs. Next we analyzed the functional consequences of these results. Addition of 1,25(OH)2D3 at concentrations comparable with those produced by DCs inhibited the allostimulatory potential of DCs during the early phase of DC differentiation. However, terminal differentiation decreased the responsiveness of DCs to 1,25(OH)2D3. In conclusion, DCs are able to produce 1,25(OH)2D3 especially following stimulation with LPS. Terminal maturation renders DCs unresponsive to the effects of 1,25(OH)2D3, but those cells are able to suppress the differentiation of their own precursor cells in a paracrine way through the production of 1,25(OH)2D3.

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Lanostane Triterpenes from the Fruiting Bodies ofGanoderma lucidumand Their Inhibitory Effects on Adipocyte Differentiation in 3T3-L1 Cells

Journal of Natural Products ◽

10.1021/np900578h ◽

2010 ◽

Vol 73 (2) ◽

pp. 172-176 ◽

Cited By ~ 35

Author(s):

IkSoo Lee ◽

JungJu Seo ◽

JinPyo Kim ◽

HongJin Kim ◽

UiJung Youn ◽

...

Keyword(s):

Adipocyte Differentiation ◽

Fruiting Bodies ◽

Inhibitory Effects

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Interrupted crosstalk between natural killer cells and anti-epidermal growth factor receptor: a possible role in hepatocellular carcinoma treatment failure

Current Cancer Drug Targets ◽

10.2174/1568009621666210519105203 ◽

2021 ◽

Vol 21 ◽

Author(s):

Hadeer Abosalema ◽

Shahenda Mahgoub ◽

Mohamed Emara ◽

Nahla Kotb ◽

Sameh Soror

Keyword(s):

Hepatocellular Carcinoma ◽

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Growth Factor Receptor ◽

Major Health Problem ◽

Advanced Stages ◽

Epidermal Growth ◽

First Time ◽

Late Stages

: Hepatocellular carcinoma (HCC) is a major health problem worldwide. Most patients are diagnosed for the first time at late stages; this leads to a very poor prognosis. It is challenging to discover strategies for treatment at these advanced stages. Recently, monoclonal antibodies (mAbs) targeting specific cellular signaling pathways in HCC have been developed. Unfortunately, they still have a low survival rate, and some of them failed clinically to produce effective responses even if they showed very good results against HCC in preclinical studies. This review focuses on and discusses the possible causes for the failure of mAbs, precisely anti-Epidermal Growth Factor Receptor (EGFR) mAb and the crosstalk between this mAb and patients' NK cells.

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