Proteomic and genomic analysis of acid dentin lysate with focus on TGF-β signaling

AbstractParticulate autologous tooth roots are increasingly used for alveolar bone augmentation; however, the proteomic profile of acid dentin lysate and the respective cellular response have not been investigated. Here we show that TGF-β1 is among the 226 proteins of acid dentin lysate (ADL) prepared from porcine teeth. RNA sequencing identified 231 strongly regulated genes when gingival fibroblasts were exposed to ADL. Out of these genes, about one third required activation of the TGF-β receptor type I kinase including interleukin 11 (IL11) and NADPH oxidase 4 (NOX4). Reverse transcription-quantitative polymerase chain reaction and immunoassay confirmed the TGF-β-dependent expression of IL11 and NOX4. The activation of canonical TGF-β signaling by ADL was further confirmed by the phosphorylation of Smad3 and translocation of Smad2/3, using Western blot and immunofluorescence staining, respectively. Finally, we showed that TGF-β activity released from dentin by acid lysis adsorbs to titanium and collagen membranes. These findings suggest that dentin particles are a rich source of TGF-β causing a major response of gingival fibroblasts.

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TGF-β Activity Related to the use of Collagen Membranes: In Vitro Bioassays

International Journal of Molecular Sciences ◽

10.3390/ijms21186636 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6636 ◽

Cited By ~ 2

Author(s):

Layla Panahipour ◽

Zahra Kargarpour ◽

Bernadette Luza ◽

Jung-Seok Lee ◽

Reinhard Gruber

Keyword(s):

Bone Regeneration ◽

Nuclear Localization ◽

Tissue Regeneration ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Guided Bone Regeneration ◽

Gingival Fibroblasts ◽

Nadph Oxidase 4 ◽

Collagen Membranes ◽

Β Receptor

Collagen membranes commonly used in guided bone regeneration are supposed to actively influence tissue regeneration and are not exclusively serving as passive barriers shielding away the soft tissue. The molecular mechanisms by which collagen membranes might affect tissue regeneration might involve the activation of transforming growth factor beta (TGF-β) signaling pathways. Here, we determined the TGF-β activity of supernatants and proteolytic lysates of five commercially available collagen membranes. The expression of TGF-β target genes interleukin 11 (IL11), NADPH oxidase 4 (NOX4), and proteoglycan 4 (PRG4) was evaluated by reverse transcriptase polymerase chain reaction and IL11 immunoassay in gingival fibroblasts. TGF-β signaling activation was further assessed by blocking the TGF-β receptor I kinase, a TGF-β neutralizing antibody, and showing the nuclear localization of phosphorylated Smad3 and total Smad2/3. We could identify two collagen membranes whose supernatants and lysates caused a robust increase of TGF-β receptor I kinase-dependent expression of IL11 in gingival fibroblasts. Moreover, the supernatant of a particular one membrane caused the nuclear localization of phosphorylated Smad3 and Smad2/3 in the fibroblasts. These results strengthen the evidence that some collagen membranes possess an intrinsic TGF-β activity that might actively influence the process of guided bone regeneration.

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RNAseq of TGF-β receptor type I kinase-dependent genes in oral fibroblast exposed to milk

BMC Oral Health ◽

10.1186/s12903-021-01913-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Layla Panahipour ◽

Dariush Mehdipour Moghaddam ◽

Jila Nasirzade ◽

Zahra Kargarpour ◽

Reinhard Gruber

Keyword(s):

Human Milk ◽

Nuclear Translocation ◽

Receptor Type ◽

Type I ◽

Gingival Fibroblasts ◽

Aqueous Fraction ◽

Tumor Necrosis Factor Ligand ◽

Oral Tissue ◽

Β Receptor ◽

Smad 3

Abstract Background Milk is a rich source of natural growth factors that may support oral tissue homeostasis and wound healing. We had shown earlier that blocking TGF-β receptor type I kinase with the inhibitor SB431542 abolished the expression of IL11 and other genes in human gingival fibroblasts exposed to the aqueous fraction of milk. Our aim was to identify the entire signature of TGF-β receptor type I kinase-dependent genes regulated by the aqueous fraction of human milk. Result RNAseq revealed 99 genes being strongly regulated by milk requiring activation of the SB431542-dependent TGF-β receptor type I kinase. Among the SB431542-dependent genes is IL11 but also cadherins, claudins, collagens, potassium channels, keratins, solute carrier family proteins, transcription factors, transmembrane proteins, tumor necrosis factor ligand superfamily members, and tetraspanin family members. When focusing on our candidate gene, we could identify D609 to suppress IL11 expression, independent of phospholipase C, sphinosine-1 phosphate synthesis, and Smad-3 phosphorylation and its nuclear translocation. In contrast, genistein and blocking phosphoinositide 3-kinases by wortmannin and LY294002 increased the milk-induced IL11 expression in gingival fibroblasts. Conclusion Taken together, our data revealed TGF-β receptor type I kinase signaling to cause major changes of the genetic signature of gingival fibroblasts exposed to aqueous fraction of human milk.

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Mechanisms of Transforming Growth Factor-β Receptor Endocytosis and Intracellular Sorting Differ between Fibroblasts and Epithelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.12.3.675 ◽

2001 ◽

Vol 12 (3) ◽

pp. 675-684 ◽

Cited By ~ 38

Author(s):

Jules J.E. Doré ◽

Diying Yao ◽

Maryanne Edens ◽

Nandor Garamszegi ◽

Elizabeth L. Sholl ◽

...

Keyword(s):

Growth Factor ◽

Epithelial Cells ◽

Ligand Binding ◽

Transforming Growth Factor ◽

Point Mutations ◽

Type I ◽

Receptor Complex ◽

Type Ii ◽

Down Regulation ◽

Β Receptor

Transforming growth factor-βs (TGF-β) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-β type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-β receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling.

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Expression of Cannabinoid Receptors in Myometrium and its Correlation With Dysmenorrhea in Adenomyosis

Reproductive Sciences ◽

10.1177/1933719119833483 ◽

2019 ◽

Vol 26 (12) ◽

pp. 1618-1625 ◽

Cited By ~ 3

Author(s):

Xue Shen ◽

Hua Duan ◽

Sha Wang ◽

Wei Hong ◽

Yu-Yan Wang ◽

...

Keyword(s):

Messenger Rna ◽

Cannabinoid Receptors ◽

Pain Perception ◽

Cannabinoid Receptor ◽

Quantitative Polymerase Chain Reaction ◽

Premenopausal Women ◽

Type I ◽

Junctional Zone ◽

Tissue Samples ◽

Expression Levels

The myometrium, especially the junctional zone (JZ), is now well documented to have a role in the pathogenesis of adenomyosis. Cannabinoid receptors have been shown to participate in the establishment of endometriosis and its pain perception. However, its relation to adenomyosis has not been identified yet. The aim of this study was to investigate the expression of cannabinoid receptor type I (CB1) and type II (CB2) in myometrium of uteri with and without adenomyosis and determine the correlation between their levels and clinical parameters of adenomyosis. We collected tissue samples of JZ and the outer myometrium from 45 premenopausal women with adenomyosis and 34 women without adenomyosis. CB1 and CB2 messenger RNA (mRNA) and protein expression levels were evaluated by the use of Western blotting and real-time quantitative polymerase chain reaction from all samples. Clinical information on the severity of dysmenorrhea and other data were collected. We found both CB1 and CB2 mRNA and protein levels in women with adenomyosis were significantly higher than those of controls, and CB1 expression levels in JZ were positively correlated with the severity of dysmenorrhea. These data suggest that cannabinoid receptor CB1 may be involved in the pathogenesis of dysmenorrhea in adenomyosis and may be a potential therapeutic target.

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Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis

The Journal of Cell Biology ◽

10.1083/jcb.200706034 ◽

2007 ◽

Vol 179 (5) ◽

pp. 935-950 ◽

Cited By ~ 100

Author(s):

K.G. Suresh Kumar ◽

Hervé Barriere ◽

Christopher J. Carbone ◽

Jianghuai Liu ◽

Gayathri Swaminathan ◽

...

Keyword(s):

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Interferon Α ◽

Type I ◽

Lysosomal Degradation ◽

Linear Motif ◽

Site Specific ◽

Receptor Endocytosis ◽

Β Receptor

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.

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Reversible ubiquitination regulates the Smad/TGF-β signalling pathway

Biochemical Society Transactions ◽

10.1042/bst0340761 ◽

2006 ◽

Vol 34 (5) ◽

pp. 761-763 ◽

Cited By ~ 40

Author(s):

S.J. Wicks ◽

T. Grocott ◽

K. Haros ◽

M. Maillard ◽

P. ten Dijke ◽

...

Keyword(s):

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Type I ◽

Serine Threonine Kinase ◽

Full Spectrum ◽

Threonine Kinase ◽

Pathological Conditions ◽

C Terminus ◽

Β Receptor ◽

Fine Tune

TGF-β (transforming growth factor-β) signals through serine/threonine kinase receptors and intracellular Smad transcription factors. An important regulatory step involves specific ubiquitination by Smurfs (Smad–ubiquitin regulatory factors), members of the HECT (homologous to E6-associated protein C-terminus) ubiquitin ligase family, which mediate the proteasomal degradation of Smads and/or receptors. Recently, we have defined a novel interaction between Smads and UCH37 (ubiquitin C-terminal hydrolase 37), a DUB (de-ubiquitinating enzyme) that could potentially counteract Smurf-mediated ubiquitination. We have demonstrated specific interactions between UCH37 and inhibitory Smad7, as well as weaker associations with Smad2 and Smad3. Importantly, Smad7 can act as an adaptor able to recruit UCH37 to the type I TGF-β receptor. Consequently, UCH37 dramatically up-regulates TGF-β-dependent gene expression by de-ubiquitinating and stabilizing the type I TGF-β receptor. Our findings suggest that competing effects of ubiquitin ligases and DUBs in complex with Smad7 can serve to fine-tune responses to TGF-βs under various physiological and pathological conditions. Studies are currently under way using activity-based HA (haemagglutinin)-tagged ubiquitin probes to identify the full spectrum of DUBs that impact on Smad/TGF-β signalling activity.

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The type I TGF-β receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner

Nature Cell Biology ◽

10.1038/ncb1780 ◽

2008 ◽

Vol 10 (10) ◽

pp. 1199-1207 ◽

Cited By ~ 360

Author(s):

Alessandro Sorrentino ◽

Noopur Thakur ◽

Susanne Grimsby ◽

Anders Marcusson ◽

Verena von Bulow ◽

...

Keyword(s):

Type I ◽

Receptor Kinase ◽

Independent Manner ◽

Β Receptor

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A Novel Protein Distinguishes between Quiescent and Activated Forms of the Type I Transforming Growth Factor β Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.273.16.9365 ◽

1998 ◽

Vol 273 (16) ◽

pp. 9365-9368 ◽

Cited By ~ 46

Author(s):

Min-Ji Charng ◽

Dou Zhang ◽

Paı̈vi Kinnunen ◽

Michael D. Schneider

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Type I ◽

Β Receptor ◽

Novel Protein

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P029 TRIM 21 protects against ulcerative colitis and colitic cancer via smad7 inhibition

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjz203.158 ◽

2020 ◽

Vol 14 (Supplement_1) ◽

pp. S146-S146

Author(s):

K B Hahm

Keyword(s):

Ulcerative Colitis ◽

Coiled Coil ◽

Type I ◽

Typical Structure ◽

Th17 Cell Differentiation ◽

Bowel Diseases ◽

Colitic Cancer ◽

Β Receptor ◽

Trim Proteins

Abstract Background Proteins of the tripartite motif-containing (TRIM) superfamily are critical in a variety of biological processes in either innate immunity or eliminating invading pathogens, by which had been implicated in pathogenesis of autoimmune diseases including inflammatory bowel diseases. The typical structure of TRIM proteins contains a RING motif in the N-terminal end, followed by a B-box motif, a coiled-coil domain and a B30.2/PRYSPRY region in the C-terminal end led to the regulation of TGF-β anti-inflammatory cytokines, by which TRIM21 has been reported to regulate IBD negatively through inhibiting Th1/Th17 cell differentiation. Methods Since antisense oligonucleotide targeting smad7 was withdrawn from clinical trial due to insufficient efficacy, in this study, we generated TRIM21 overexpressed cell lines to study the binding of TRIM21 to smad7 as well as the regulation of consequent TGF-β receptor. Results TRIM21 significantly binds to smad7 as well as repressed levels of TGF-b type I/II receptor. SBE-luc and 3TP-luc assay showed significantly decreased activities under TRIM21 + TGF-β. Since TRIM21 contains ubiquitin ligase, PRYSPRY, TRIM21 with TGF-β significantly decreased TGFRII via UPL. These in vitro evidences that TRIM21 significantly repressed TGF-β after binding smad7 were validated with DSS-induced colitis and colitic cancer model. TRIM21 was significantly decreased in DSS-induced ulcerative colitis, whereas ameliorated colitis showed significant restoration of TRIM21 Conclusion Leading to conclusion that loss of TRIM21 led to significant bout of IBD.

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