Pref-1 Interacts with Fibronectin To Inhibit Adipocyte Differentiation

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.02207-06 ◽

2006 ◽

Vol 27 (6) ◽

pp. 2294-2308 ◽

Cited By ~ 116

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.

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Ectodomain Shedding of Preadipocyte Factor 1 (Pref-1) by Tumor Necrosis Factor Alpha Converting Enzyme (TACE) and Inhibition of Adipocyte Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.02437-05 ◽

2006 ◽

Vol 26 (14) ◽

pp. 5421-5435 ◽

Cited By ~ 94

Author(s):

Yuhui Wang ◽

Hei Sook Sul

Keyword(s):

Tumor Necrosis Factor ◽

Tumor Necrosis Factor Alpha ◽

Tumor Necrosis ◽

Adipocyte Differentiation ◽

Soluble Form ◽

Dependent Manner ◽

Converting Enzyme ◽

Necrosis Factor Alpha ◽

Factor Alpha ◽

Necrosis Factor

ABSTRACT Preadipocyte factor 1 (Pref-1), an epidermal growth factor repeat containing transmembrane protein found in the preadipocytes, inhibits adipocyte differentiation in vitro and in vivo. Here, we examined the processing of membrane form of Pref-1A to release the 50-kDa soluble form that inhibits adipocyte differentiation. The ectodomain cleavage of Pref-1 is markedly enhanced by phorbol 12-myristate 13-acetate in a dose- and time-dependent manner. The basal and stimulated cleavage is inhibited by the broad metalloproteinase inhibitor GM6001, a fact that suggests that cleavage of membrane Pref-1A is dependent on a metalloproteinase. Next, we showed that release of soluble Pref-1A is inhibited by TAPI-0 and by a tissue inhibitor of metalloproteinase-3, TIMP-3, that can inhibit tumor necrosis factor alpha converting enzyme (TACE), but not by TIMP-1 or TIMP-2. On the other hand, overexpression of TACE increases Pref-1 cleavage to produce the 50-kDa soluble form. Furthermore, this cleavage was not detected in cells with TACE mutation or with TACE small interfering RNA. TACE-mediated shedding of Pref-1 ectodomain inhibits adipocyte differentiation of 3T3-L1 cells and in Pref-1-null mouse embryo fibroblasts transduced with Pref-1A. Identification of TACE as the major protease responsible for conversion of membrane-bound Pref-1 to the biologically active diffusible form provides a new insight into Pref-1 function in adipocyte differentiation.

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Coordinate Regulation of IκB Kinases by Mitogen-Activated Protein Kinase Kinase Kinase 1 and NF-κB-Inducing Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7336 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7336-7343 ◽

Cited By ~ 181

Author(s):

Shino Nemoto ◽

Joseph A. DiDonato ◽

Anning Lin

Keyword(s):

Protein Kinase ◽

Interleukin 1 ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Dominant Negative Mutant ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

Ikk Complex ◽

Mitogen Activated Protein ◽

Protein Kinase Kinase

ABSTRACT IκB kinases (IKKα and IKKβ) are key components of the IKK complex that mediates activation of the transcription factor NF-κB in response to extracellular stimuli such as inflammatory cytokines, viral and bacterial infection, and UV irradiation. Although NF-κB-inducing kinase (NIK) interacts with and activates the IKKs, the upstream kinases for the IKKs still remain obscure. We identified mitogen-activated protein kinase kinase kinase 1 (MEKK1) as an immediate upstream kinase of the IKK complex. MEKK1 is activated by tumor necrosis factor alpha (TNF-α) and interleukin-1 and can potentiate the stimulatory effect of TNF-α on IKK and NF-κB activation. The dominant negative mutant of MEKK1, on the other hand, partially blocks activation of IKK by TNF-α. MEKK1 interacts with and stimulates the activities of both IKKα and IKKβ in transfected HeLa and COS-1 cells and directly phosphorylates the IKKs in vitro. Furthermore, MEKK1 appears to act in parallel to NIK, leading to synergistic activation of the IKK complex. The formation of the MEKK1-IKK complex versus the NIK-IKK complex may provide a molecular basis for regulation of the IKK complex by various extracellular signals.

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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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IL-1 receptor–associated kinase M is a central regulator of osteoclast differentiation and activation

Journal of Experimental Medicine ◽

10.1084/jem.20041444 ◽

2005 ◽

Vol 201 (7) ◽

pp. 1169-1177 ◽

Cited By ~ 48

Author(s):

Hongmei Li ◽

Esteban Cuartas ◽

Weiguo Cui ◽

Yongwon Choi ◽

Todd D. Crawford ◽

...

Keyword(s):

Bone Loss ◽

Bone Fractures ◽

Bone Development ◽

Osteoclast Differentiation ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Downstream Signaling ◽

Signaling Complex ◽

Mitogen Activated Protein ◽

Protein Kinase Signaling

Osteoporosis is a serious problem worldwide; it is characterized by bone fractures in response to relatively mild trauma. Osteoclasts originate from the fusion of macrophages and they play a central role in bone development and remodeling via the resorption of bone. Therefore, osteoclasts are important mediators of bone loss that leads, for example, to osteoporosis. Interleukin (IL)-1 receptor (IL-1R)–associated kinase M (IRAK-M) is only expressed in cells of the myeloid lineage and it inhibits signaling downstream of IL-1R and Toll-like receptors (TLRs). However, it lacks a functional catalytic site and, thus, cannot function as a kinase. IRAK-M associates with, and prevents the dissociation of, IRAK–IRAK-4–TNF receptor–associated factor 6 from the TLR signaling complex, with resultant disruption of downstream signaling. Thus, IRAK-M acts as a dominant negative IRAK. We show here that mice that lack IRAK-M develop severe osteoporosis, which is associated with the accelerated differentiation of osteoclasts, an increase in the half-life of osteoclasts, and their activation. Ligation of IL-1R or TLRs results in hyperactivation of NF-κB and mitogen-activated protein kinase signaling pathways, which are essential for osteoclast differentiation. Thus, IRAK-M is a key regulator of the bone loss that is due to osteoclastic resorption of bone.

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PDGF-BB-mediated activation of p42MAPK is independent of PDGF β-receptor tyrosine phosphorylation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.4.l786 ◽

2001 ◽

Vol 281 (4) ◽

pp. L786-L798 ◽

Cited By ~ 10

Author(s):

Nicholas J. Cartel ◽

Jason Liu ◽

Jinxia Wang ◽

Martin Post

Keyword(s):

Tyrosine Phosphorylation ◽

Dominant Negative ◽

Janus Kinase ◽

Mitogen Activated Protein Kinase ◽

Phosphatidylinositol 3 Kinase ◽

Downstream Signaling ◽

Mitogen Activated Protein ◽

Signaling Domain ◽

Chemical Inhibition ◽

Β Receptor

Herein, we investigated the activity of mitogen-activated protein kinase (MAPK), a key component of downstream signaling events, which is activated subsequent to platelet-derived growth factor (PDGF)-BB stimulation. Specifically, p42MAPK activity peaked 60 min after addition of PDGF-BB, declined thereafter, and was determined not to be a direct or necessary component of glycosaminoglycan (GAG) synthesis. PDGF-BB also activated MAPK kinase 2 (MAPKK2) but had no effect on MAPKK1 and Raf-1 activity. Chemical inhibition of Janus kinase, phosphatidylinositol 3-kinase, Src kinase, or tyrosine phosphorylation inhibition of the PDGF β-receptor (PDGFR-β) did not abrogate PDGF-BB-induced p42MAPK activation or its threonine or tyrosine phosphorylation. A dominant negative cytoplasmic receptor for hyaluronan-mediated motility variant 4 (RHAMMv4), a regulator of MAPKK-MAPK interaction and activation, did not inhibit PDGF-BB-induced p42MAPK activation nor did a construct expressing PDGFR-β with cytoplasmic tyrosines mutated to phenylalanine. However, overexpression of a dominant negative PDGFR-β lacking the cytoplasmic signaling domain abrogated p42MAPKactivity. These results suggest that PDGF-BB-mediated activation of p42MAPK requires the PDGFR-β but is independent of its tyrosine phosphorylation.

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Differential expression and processing of two cell associated forms of the kit-ligand: KL-1 and KL-2.

Molecular Biology of the Cell ◽

10.1091/mbc.3.3.349 ◽

1992 ◽

Vol 3 (3) ◽

pp. 349-362 ◽

Cited By ~ 207

Author(s):

E J Huang ◽

K H Nocka ◽

J Buck ◽

P Besmer

Keyword(s):

Transmembrane Protein ◽

Active Form ◽

Calcium Ionophore ◽

Proteolytic Cleavage ◽

Proteolytic Processing ◽

Biologically Active ◽

Soluble Form ◽

Predicted Amino Acid Sequence ◽

Ionophore A23187 ◽

Kit Ligand

The c-kit ligand, KL, and its receptor, the proto-oncogene c-kit are encoded, respectively, at the steel (Sl) and white spotting (W) loci of the mouse. Both Sl and W mutations affect cellular targets in melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. Although identified as a soluble protein, the predicted amino acid sequence of KL indicates that it is an integral transmembrane protein. We have investigated the relationship between the soluble and the cell associated forms of KL and the regulation of their expression. We show that the soluble form of KL is generated by efficient proteolytic cleavage from a transmembrane precursor, KL-1. An alternatively spliced version of KL-1, KL-2, in which the major proteolytic cleavage site is removed by splicing, is shown to produce a soluble biologically active form of KL as well, although with somewhat diminished efficiency. The protein kinase C inducer phorbol 12-myristate 13-acetate and the calcium ionophore A23187 were shown to induce the cleavage of both KL-1 and KL-2 at similar rates, suggesting that this process can be regulated differentially. Furthermore, proteolytic processing of both the KL-1 and KL-2 transmembrane protein products was shown to occur on the cell surface. The relative abundance of KL-1 and KL-2 is controlled in a tissue-specific manner. Sld, a viable steel allele, is shown to encode a biologically active secreted mutant KL protein. These results indicate an important function for both the soluble and the cell associate form of KL. The respective roles of the soluble and cell associated forms of KL in the proliferative and migratory functions of c-kit are discussed.

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Chrysin Inhibits TNFα-Induced TSLP Expression through Downregulation of EGR1 Expression in Keratinocytes

International Journal of Molecular Sciences ◽

10.3390/ijms22094350 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4350

Author(s):

Hyunjin Yeo ◽

Younghan Lee ◽

Sungshin Ahn ◽

Euitaek Jung ◽

Yoongho Lim ◽

...

Keyword(s):

Clinical Symptoms ◽

Skin Lesions ◽

Therapeutic Agents ◽

Mitogen Activated Protein Kinase ◽

Necrosis Factor Alpha ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

Signaling Axis ◽

Factor Alpha ◽

Necrosis Factor

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that acts as a critical mediator in the pathogenesis of atopic dermatitis (AD). Various therapeutic agents that prevent TSLP function can efficiently relieve the clinical symptoms of AD. However, the downregulation of TSLP expression by therapeutic agents remains poorly understood. In this study, we investigated the mode of action of chrysin in TSLP suppression in an AD-like inflammatory environment. We observed that the transcription factor early growth response (EGR1) contributed to the tumor necrosis factor alpha (TNFα)-induced transcription of TSLP. Chrysin attenuated TNFα-induced TSLP expression by downregulating EGR1 expression in HaCaT keratinocytes. We also showed that the oral administration of chrysin improved AD-like skin lesions in the ear and neck of BALB/c mice challenged with 2,4-dinitrochlorobenzene. We also showed that chrysin suppressed the expression of EGR1 and TSLP by inhibiting the extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2 mitogen-activated protein kinase pathways. Collectively, the findings of this study suggest that chrysin improves AD-like skin lesions, at least in part, through the downregulation of the ERK1/2 or JNK1/2-EGR1-TSLP signaling axis in keratinocytes.

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Transcriptional Regulators of theSchizosaccharomyces pombe fbp1Gene Include Two Redundant Tup1p-like Corepressors and the CCAAT Binding Factor Activation Complex

Genetics ◽

10.1093/genetics/157.3.1205 ◽

2001 ◽

Vol 157 (3) ◽

pp. 1205-1215 ◽

Cited By ~ 4

Author(s):

Rozmin T K Janoo ◽

Lori A Neely ◽

Burkhard R Braun ◽

Simon K Whitehall ◽

Charles S Hoffman

Keyword(s):

Protein Kinase ◽

Fold Increase ◽

Transcriptional Regulators ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Lacz Expression ◽

Protein Subunit ◽

Plasmid Library ◽

Binding Factor ◽

Mitogen Activated Protein

AbstractThe Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation. We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways. A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF). Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions. The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA.

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