Lee et al. (1992) described a gene, which they designated TSG6, that is transcribed in normal fibroblasts and activated by
binding of the cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL1) at AP-1
and NF-IL6 sites in its promoter. The cDNA was isolated from a library made from TNF-treated human fibroblasts. TSG6 is a
member of the hyaluronan-binding protein family, which includes cartilage link protein ), proteoglycan core protein
, and the adhesion receptor CD44.
NCBI Summary:
The protein encoded by this gene is a secretory protein that contains a hyaluronan-binding domain, and thus is a member of the hyaluronan-binding protein family. The hyaluronan-binding domain is known to be involved in extracellular matrix stability and cell migration. This protein has been shown to form a stable complex with inter-alpha-inhibitor (I alpha I), and thus enhance the serine protease inhibitory activity of I alpha I, which is important in the protease network associated with inflammation. This gene can be induced by proinflammatory cytokines such as tumor necrosis factor alpha and interleukin-1. Enhanced levels of this protein are found in the synovial fluid of patients with osteoarthritis and rheumatoid arthritis.[provided by RefSeq, Dec 2010]
General function
Ligand
Comment
Yoshioka S, et al. (Endocrinology. 2000) believed the expression of TSG-6 is an integral part of the cascade of inflammatory-like changes that occur in an ovulatory follicle in response to a trophic hormone that couples with luteinizing hormone/hCG receptors.
Cellular localization
Extracellular Matrix, Secreted
Comment
Ovarian function
Cumulus expansion, Ovulation
Comment
Inter-a-Inhibitor Impairs TSG-6-Induced Hyaluronan Cross-Linking. Baranova NS 2013 et al.
Under inflammatory conditions and in the matrix of the cumulus-oocyte complex (COC), the polysaccharide hyaluronan (HA) becomes decorated covalently with heavy chains (HCs) of the serum glycoprotein inter-a-inhibitor (IaI). This alters the functional properties of the HA as well as its structural role within extracellular matrices. The covalent transfer of HCs from IaI to HA is catalyzed by tumor necrosis factor-stimulated gene-6 (TSG-6) but TSG-6 is also known as a HA cross-linker that induces condensation of the HA matrix. Here, we investigate the interplay of these two distinct functions of TSG-6 by studying the ternary interactions of IaI and TSG-6 with well defined films of end-grafted HA chains. We demonstrate that TSG-6 mediated cross-linking of HA films is impaired in the presence of IaI, and that this effect suppresses the TSG-6-mediated enhancement of HA binding to CD44 positive cells. Furthermore, we find that the interaction of TSG-6 and IaI in the presence of HA gives rise to two types of complexes that independently promote the covalent transfer of heavy chains to HA. One type of complex interacts very weakly with HA and is likely to correspond to the previously reported covalent HCTSG-6 complexes. The other type of complex is novel and binds stably but non-covalently to HA. Prolonged incubation with TSG-6 and IaI leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but non-covalently bound molecular species. These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of IaI will dictate its function.
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Regulation of bovine tumor necrosis factor alpha induced protein 6 in ovarian follicles during the ovulatory process and promoter activation in granulosa cells. Sayasith K et al. To study the regulation of bovine tumor necrosis factor alpha induced protein 6 (TNFAIP6) prior to ovulation, preovulatory follicles obtained after the treatment with human chorionic gonadotropin (hCG) were used. RT-PCR analyses showed that levels of TNFAIP6 mRNA were low before hCG, but significant increased after hCG treatment in follicles. Further analyses and immunohistochemistry indicated that this increase in transcript and protein levels occurred in theca and granulosa cells. To investigate molecular mechanisms involved in TNFAIP6 transactivation, the activity of bovine TNFAIP6 promoter was studied in granulosa cell cultures. Mutant studies identified the minimal region conferring full-length promoter activity, in which AP1 and CRE elements were required for promoter activity. Overexpression of dominant-negative AP1 and ATF/CREB inhibited foskolin-inducible promoter activity. DNA binding assays demonstrated the importance of AP1 and CRE for activity, and identified JunD, FosB, Fra2, CREB1, and CREB2 being part of the AP1-complex, and FosB, Fra2 and CREB1 for the CRE-complex. Chromatin immunoprecipitation assays confirmed binding of these proteins with endogenous TNFAIP6 promoter. Treatment with forskolin, prostaglandin E2 and catalytic subunit protein kinase (cPKA) stimulated, but H89, PKA inhibitor peptide and indomethacin inhibited, TNFAIP6 promoter activity and gene expression in granulosa cells. Collectively, this study is the first to describe that the ovulatory process in cows is associated with a gonadotropin-dependent induction of TNFAIP6 in ovarian follicles, and to provide the molecular basis through which AP1 and CRE sites and PKA activation played important roles in the regulation of TNFAIP6 in granulosa cells.
Ochsner SA, et al reported disrupted function of TNF-{alpha} stimulated gene 6 blocks cumulus cell-oocyte complex expansion. TSG-6 mRNA is induced in cumulus cells in culture by cAMP and that the secreted TSG-6 protein is a key structural component of the mouse COC matrix. Ochsner SA, et al.(2003)
identified TSG-6 as a target of PG action and show that its production in ovulatory follicles is associated with proper formation of the cumulus-derived extracellular matrix. IN situ hybridization indicated that TSG^ is expressed in both mural and cumulus granulosa cells of antral follicles. This message showed the highest levels at 6 h after hCG treatment of preovulatory follicles.
Mukhopadhyay D et al reported the specificity of the tumor necrosis factor-induced protein 6-mediated heavy chain transfer from inter-alpha -trypsin inhibitor to hyaluronan. Implications for the assembly of the cumulus extracellular matrix.
Expression regulated by
LH
Comment
Molecular characterization of tumor necrosis {alpha}-induced protein 6 and its human chorionic gonadotropin-dependent induction in theca and mural granulosa cells of equine preovulatory follicles. Sayasith K et al. The preovulatory rise in gonadotropins causes an expansion of the cumulus-oocyte complex, a process requiring the induction of several genes. The objectives of this study were to clone the equine tumor necrosis factor alpha-induced protein 6 (TNFAIP6), and investigate its regulation in equine follicles during human chorionic gonadotropin (hCG)-induced ovulation. The isolation of the equine TNFAIP6 cDNA revealed that it contains an open reading frame of 834 bp (including the stop codon), encoding a predicted 277 amino acid protein that is highly similar (91-93% identity) to known mammalian homologs. The regulation of TNFAIP6 mRNA was studied in equine follicles isolated during estrus between 0 and 39 h post-hCG and in corpora lutea (CL) obtained on day 8 of the estrous cycle. Results from semi-quantitative RT-PCR/Southern blot showed that levels of TNFAIP6 mRNA were low in follicles obtained at 0 h, increased at 12 h, returned to basal levels at 24 h, and increased again at 36 h post-hCG (P<0.05). Levels of TNFAIP6 transcripts were relatively moderate in CL, but low in non-ovarian tissues tested. Analyses performed with isolated preparations of theca and granulosa cells indicated that TNFAIP6 mRNA was regulated in both layers, with a maximal induction obtained 33-36 h post-hCG (P<0.05). Immunohistochemical staining of sections of equine follicles isolated at 0 and 33 h post-hCG confirmed the induction of TNFAIP6 protein in both cell types after hCG treatment. Thus, the present study describes for the first time the gonadotropin-dependent regulation of follicular TNFAIP6 during the ovulation in a monoovulatory species. The biphasic induction of TNFAIP6 in equine theca and granulosa cells differs from the pattern observed in rodents, suggesting a distinct control of gene expression in this monoovulatory species.
Ovarian localization
Cumulus, Granulosa, Theca, Luteal cells
Comment
Progressive changes in human follicular fluid composition over the course of ovulation: Quantitative proteomic analyses. la Cour Poulsen L et al. (2019) Follicular fluid (FF) acts as a vehicle for paracrine signalling between somatic cells of the follicle and the oocyte. To investigate changes in the protein composition of FF during ovulation, we conducted a prospective cohort study including 25 women undergoing fertility treatment. Follicular fluid was aspirated either before or 12, 17, 32 or 36 h after induction of ovulation (five patients per time point). Liquid chromatography-mass spectrometry was used to identify and quantify FF proteins. In total, 400 proteins were identified and the levels of 40 proteins changed significantly across ovulation evaluated by analysis of covariance (adjusted p < 0.05) and on-off expression patterns. The majority peaked after 12-17 h, e.g., AREG (p < 0.0001), TNFAIP6 (p < 0.0001), and LDHB (p = 0.0316), while some increased to peak after 36 h e.g., ACPP (p < 0.0001), TIMP1 (p < 0.0001) and SERPINE1 (p = 0.0002). Collectively, this study highlights proteins and pathways of importance for ovulation and oocyte competence in humans.//////////////////
Odile Carrette et al reported that TSG-6 is concentrated in the extracellular matrix of
mouse cumulus oocyte complexes through hyaluronan
and inter-alpha-inhibitor binding.
During development of ovarian follicles in mammals, cumulus cells and the
oocyte form a mucoelastic mass that detaches itself from peripheral granulosa
cell layers upon an ovulatory surge. The integrity of this cumulus-oocyte
complex (COC) relies on the cohesiveness of a hyaluronan (HA)-enriched
extracellular matrix (ECM). The authors previously identified a serum glycoprotein,
inter-alpha-inhibitor (IalphaI), that is critical in organizing and stabilizing this
matrix. Following an ovulatory stimulus, IalphaI diffuses into the follicular fluid
and becomes integrated in the ECM through its association with HA. TSG-6
(the secreted product of the tumor necrosis factor-stimulated gene 6),
another HA binding protein, forms a complex with IalphaI in synovial fluid.
Immunolocalization of TSG-6 and IalphaI in
mouse COCs at different ovulatory stages was analyzed by
immunofluorescence and laser confocal microscopy. IalphaI, TSG-6, and
HA colocolized in the cumulus ECM. Western blot analyses were consistent
with the presence of both TSG-6 and TSG-6/IalphaI complexes in ovulated
COCs. These results suggest that TSG-6 has a structural role in COC matrix
formation possibly mediating cross-linking of separate HA molecules through
its binding to IalphaI.
Follicle stages
Antral, Preovulatory, Corpus luteum
Comment
By analyzing GDF-9-regulated expression profiles using gene chip technology, the authors identified TNF-induced protein 6 (Tnfip6) and pentraxin 3 (Ptx3 or PTX3) as novel factors induced by GDF-9 in granulosa cells of preovulatory follicles. Whereas Tnfip6 is induced in all granulosa cells by the LH surge, Ptx3 expression in the ovary is specifically observed after the LH surge in the cumulus granulosa cells adjacent to the oocyte. PTX3 is a member of the pentraxin family of secreted proteins, induced in several tissues by inflammatory signals.(Varani S, et al )
Synthesis of Tumor Necrosis Factor Alpha-Induced Protein 6 in Porcine Preovulatory Follicles: A Study with A38 Antibody. Nagyova E et al. We have previously shown that the heavy chains (HC) of inter-alpha-trypsin inhibitor (IalphaI) become covalently linked to hyaluronan (HA) during in vivo and in vitro expansion of porcine oocyte-cumulus cell complexes (OCCs). We have now studied by immunoblotting the synthesis of tumor necrosis factor alpha induced protein 6 (TNFAIP6), which is essential for catalyzing this reaction in expanding mouse OCCs. Expanding OCCs were collected from preovulatory follicles of naturally cycling pigs and also after in vitro culture (24 or 42 h) in medium supplemented with FSH and pig serum. After isolation, OCCs were treated with Streptomyces hyaluronidase or Chondroitinase ABC. Matrix, cell pellet and total extracts were analyzed by Western blotting. A band of about 35-kDa and a doublet of about 120-kDa, corresponding to the molecular weight of the native and HC-linked form of TNFAIP6, respectively, were detected by a rabbit anti-human TNFAIP6 polyclonal antibody in matrix extracts of expanded cumuli. Moreover, we found, by using a cell-free assay that porcine follicular fluid collected from follicles at 24 h after hCG stimulation contains HC-HA coupling activity. This activity was abolished by the rat anti-human monoclonal antibody A38, which has an epitope within the Link module domain of TNFAIP6. These experiments suggest that free TNFAIP6 protein was present in follicular fluid aspirated from porcine follicles 24 h after hCG stimulation. In contrast to mouse, we show that the A38 monoclonal antibody does not affect in vitro cumulus expansion of porcine OCCs.
Phenotypes
Mutations
1 mutations
Species: mouse
Mutation name: None
type: null mutation fertility: infertile - ovarian defect Comment:Fulop C et al reported impaired cumulus mucification and female sterility in tumor necrosis
factor-induced protein-6 deficient mice.
Mucification of the cumulus layer around the oocyte is an obligatory process for female fertility.
Tumor necrosis factor-induced protein-6 (TNFIP6 or TSG6) has been shown to be specifically
expressed during this process. Cumulus cell-oocyte complexes fail to expand in
TNFIP6-deficient female mice because of the inability of the cumulus cells to assemble their
hyaluronan-rich extracellular matrix. The impaired cumulus matrix formation is due to the lack of
covalent complexes between hyaluronan and the heavy chains of the inter-alpha-trypsin inhibitor
family. As a consequence, TNFIP6-deficient females are sterile. Cultured TNFIP6-deficient cumulus
cell-oocyte complexes also fail to expand when stimulated with dibutyryl cyclic AMP or epidermal
growth factor. Recombinant TNFIP6 is able to catalyze the covalent transfer of heavy chains to
hyaluronan in a cell-free system, restore the expansion of Tnfip6-null cumulus cell-oocyte
complexes in vitro, and rescue the fertility in Tnfip6-null females. These results provide clear
evidence that TNFIP6 is a key catalyst in the formation of the cumulus extracellular matrix and
indispensable for female fertility.