Overexpressed nephroblastoma protein - a review (2023)

2.1 CCN-Protein

Taken together, mammalian CCNs play multiple developmental roles in cartilage, bone, and other organs. They promote angiogenesis and are elevated in many disease processes, including wound healing, inflammation, fibrosis, and cancer.Hutchenreuther, Leask & Thompson, 2017;Krupska, Bruford and Chaqour, 2015). In particular, CCN2 has an advantageous effect as a driver of myofibroblast differentiation in various tissues.Hall-Glenn & Lyons, 2011;Leask, 2013;Maurer, 2013). Unlike the other families of mesenchymal genes, there are severalccnHowever, gene knockout mice display profound developmental phenotypesccn3mccn4Gene knockout mice are viable. For this reason,ccn1^−/−Mice die during embryogenesis due to failure of vascular development resulting in hypovascularization of the placenta.Mo et al., 2002).Ctfg/ccn2^−/−Mice die at birth with defective development of pancreatic islet organs, skeleton and palate and respiratory failure.Crawford et al., 2009;Ivkovic et al., 2003;Lampi et al., 2012;Tarr et al., 2017).Cnn5^−/−Mice die during embryogenesis during germline implantation (Myers, Fourth, Richey, Lem Castellot, 2012). Specific knockout of the mammary glandccn6leads to an increased incidence of invasive breast carcinomas.Martin et al., 2017). Point mutations occur in humansCCN6associated with progressive pseudorheumatoid dysplasia (Table 1).

CCNs associate with other ECM proteins and bind to growth factors, cytokines, and a variety of cell surface receptors including αβ3 and α5β1 integrins and heparan sulfate proteoglycans as integrin co-receptors.Lau, 2016). As discussed in detail byLau (2016)There are numerous additional receptors for CCN2 on different cells, including insulin-like growth factor (IGF)-2 receptor, LDL receptor-related protein-1 (LRP-1), TrkA, growth factor receptor-2 fibroblasts and factor κB receptor activator and specific dendritic cell transmembrane protein. Notch-1 has been identified as a receptor for CCN3 (Wagener, Yang, Kazuschke, Winterhager & Gelhaus, 2013).

4.5.6 CCN

The CCN family consists of cysteine-rich protein 61 (CCN1), connective tissue growth factor (CTGF, CCN2),overexpressed nephroblastoma protein(CCN3) and the Wnt-inducible secreted proteins CCN4, CCN5 and CCN6. CCN proteins regulate cell adhesion, migration and survival. CCN1, CCN2 and CCN3 promote the migration of inflammatory cells, endothelial cells and fibroblasts, while CCN4 and CCN5 inhibit their migration.71,72]. CCN1 induces the proinflammatory polarization of macrophages and promotes the expression of genes responsible for adhesion, angiogenesis, and ECM renewal.73]. CCN1 is strongly induced in end-stage ischemic cardiomyopathy and regulates apoptosis, angiogenesis, inflammation and fibrotic response.74,75]. In an ischemia reperfusion model, overexpression of CCN2 in cardiomyocytes reduces infarct size by modulating Akt/p70S6/GSK-3β kinase signaling [76].in vitro, CCN4 stimulates cardiomyocyte hypertrophy, inhibits cardiomyocyte apoptosis, and facilitates cardiac fibroblast proliferation.77]. After myocardial infarction, CCN4 induction peaks at the infarct tip and distal regions at 24 h [77]. Actions related to MI by other CCN members have not been investigated.

2.3 Genetic modification of cells

Because it is difficult to obtain enough cells from vitiligo patients to test all hypotheses, genetic data suggest that we can modify sensitive genes using overexpression or silencing vectors, such as nephroblastoma overexpression protein (NOV), also known as CCN3 (homeostasis ). melanocyte adhesion), discoidin receptor 1 (DDR1) (melanocyte adhesion to basement membrane) and cadherins (cell adhesion)[20,21]. Depending on the time of experiment or laboratory agreement, two types of vectors can be used in the cells. For a short time, transient modification (transfection) studies are performed using a plasmid encoding the gene of interest or siRNA.[20]. However, long-term studies require viral infection (transduction) using viral particles. Different types of viruses encoding the gene of interest or shRNA can be used. Viruses (retroviruses, lentiviruses, adenoviruses, or adenoviruses (AAV)) differ primarily in their ability to infect proliferating or quiescent cells and to integrate or not into the host genome. To circumvent the fact that shRNA does not cause 100% inhibition, CRISPR technology was developed. Another important point, especially in long-term studies, is the decision whether to express or suppress the gene permanently or at a specific point in time. In the latter case, inducible promoters or tet-on/tet-off technologies are used.

After the first or second passage, cells are incubated with virus particles at a multiplicity of infection (MOI) of 10 in a small volume of serum-free medium. After 6-16 hours, fresh medium containing FCS is added to bring the volume to normal (eg to 25 cm).²Transduction takes place in 0.5 ml of serum-free medium and then 3 ml of medium containing FCS is added. The medium is changed after 48 h and the percentage of transduction using a fluorescent reporter gene (eg, RFP, GFP, and YFP) present in the vector is estimated 72 h after transduction to avoid overestimation due to pseudoconversion. To achieve 100% transduction, an antibiotic resistance gene, such as the puromycin resistance gene, can be added to the vector construct. Alternatively, cells can be sorted by flow cytometry to achieve approximately 95% transduction.

Genes associated with CHS

Aldehyde dehydrogenase (ALDH) has been reported to be a key regulator of HSC differentiation[33]. Inhibition of ALDH with diethylaminobenzaldehyde (DEAB) delayed the differentiation of human HSCs that would otherwise occur in response to cytokines. In addition, short-term culture with DEAB induced a 3.4-fold increase in HSC[33]. The effects of DEAB on HSC differentiation can be reversed by co-administration of the retinoic acid receptor agonist all-trans retinoic acid, suggesting that the ability of ALDH to produce retinoic acids is important in determining the fate of HSCs. High levels of the ALDH1A1 isoform of aldehyde dehydrogenase are expressed in HSCs, but loss of ALDH1A1 does not affect them[34]. ALDH1A1 deficiency is associated with increased expression of the ALDH3A1 isoform, suggesting that it may balance ALDH1A1. ALDH3A1 and ALDH1A1-deficient mice exhibit reduced HSC numbers and abnormal cell cycle distribution, increased levels of reactive oxygen species, p38 mitogen-activated protein kinase activity, and susceptibility to DNA damage[34]. These results demonstrate that ALDH3A1 can compensate for ALDH1A1 and that both ALDH1A1 and 3A1 genes are important in HSC metabolism of reactive oxygen species and reactive aldehydes.

Matricellular protein Nephroblastoma Overexpressed (Nov, CCN3) is essential for HSC functional integrity[35]. Forced expression of Nov or addition of recombinant Nov protein increases HSC and/or progenitor activity. Scl and Lyl1 encode two related basic helix-loop-helix transcription factors, and single- and double-knockout studies in mice have shown that the expression of at least one of these factors is necessary to maintain HSC function in adults[36]. Lineage-related transcription factors must be tightly regulated in HSC self-renewal and commitment decisions. During embryogenesis, Runx1/AML1 and Evi-1 are essential for HSCs, while in adults Runx1 and Evi-1 up- and down-regulate HSCs, respectively (see ref.).[37]). The tumor suppressor and cell contact inhibitory mediator Nf2/Merlin is critical for maintaining the normal structure and function of the HSC niche[38]. HSCs in Nf2-deficient mice were increased in number and exhibited a marked change in circulatory location, which was associated with increases in vascular endothelial growth factor (VEGF) and bone and vascular components[38]. Nf2 demarcates the bony and vascular components of the niche and thus restricts HSC number and location. Disruption of the c-myb proto-oncogene specifically in the adult bone marrow resulted in critical depletion of the HSC pool with impaired hematopoiesis and a severe reduction in neutrophil, monocyte, B-lymphoid, erythroid, and megakaryocytic cell numbers[39]. This transcription factor is an essential regulator of self-renewal and multilineage differentiation of long- and short-term adult HSCs and pluripotent progenitors. Co-expression of lymphoid and myeloid genes is an early event that occurs in pluripotent progenitors but also in approximately 30% of short-lived HSCs[40].

Proliferative nuclear antigen-associated factor (PAF) is highly expressed in the BM-HSC cycle and plays a key role as a key regulator of early hematopoiesis[41]. Mice lacking Paf showed reduced BM cellularity and leukopenia with reduced numbers of HSCs and impaired progenitors. These phenotypes are caused by an intrinsic cell block in the development of long-term HSCs (LT) into pluripotent progenitors and a preferential loss of lymphoid progenitors caused by significantly increased p53-mediated apoptosis[41]. In addition, LT-HSCs from Paf(−/−) mice displayed increased levels of reactive oxygen species (ROS), failed to maintain quiescence, and were unable to support long-term hematopoiesis.

Focal adhesion kinase (Fak) is a receptor-free protein tyrosine kinase that plays an essential role in many cell types, where its activation controls adhesion, motility and survival. Fak expression is relatively elevated in HSCs compared to progenitor cells and mature blood cells[42]. Extensive Fak inactivation in the hematopoietic system leads to an increase in the number of activated HSCs, likely due to changes in the mutual interactions between HSCs and their microenvironment[42]. It has been reported that the endoplasmic reticulum chaperone protein GRP94 is essential for the expression of specific integrins and is required to maintain HSC interactions with the BM site in adults[43]. There was an increase in HSCsand granulocyte-monocyte precursors in Grp94^−/−BM is associated with loss of HSC quiescence and increased proliferation. This expansion of the HSC pool may be attributed to the impaired interaction of HSCs with their niche, indicated by severely reduced HSC homing and homing potential, as well as increased mobilization[43].

Endoglin is an accessory transforming growth factor-beta (TGF-beta) receptor that has recently been shown to be highly expressed in long-term repopulated HSCs[44]. However, little is known about its function in these cells. Transplantation of BM enriched with HSCs and progenitor cells revealed that neither endoglin suppression nor endoglin overexpression affected the ability of stem cells to repopulate recipient marrow in the short or long term[44]. Reduced endoglin expression increased the clonogenic capacity of erythroid precursors of the blastocyst, while overexpression reduced erythroid differentiation to the basophilic erythroblast phase, suggesting that endoglin plays a key role in key stages of adult erythropoietic development[44].

Protein regulation through the ubiquitin-proteasome system (UPS) has recently been shown to be critical for normal HSC function (see ref.).[26]). Casita B-cell lymphoma (c-Cbl), Itch and Fbw-7 reported as the 'sleeping guardians' of the HSC population[26]. The c-Cbl proto-oncogene is an E3-finger ubiquitin ligase that is the cellular homologue of v-Cbl, a retroviral transformant c-Cbl gene that directly or indirectly regulates approximately 150 proteins and regulates Notch1 activity downregulates , c- Kit and Signal Transducer and Transcriptional Activator (STAT)5 (activator of c-myc expression), which contribute to the maintenance of HSC[26]. Cbl knockout mice show cell-autonomous growth in HSCs without an increase in mature cell production. cbl^−/−HSCs show increased levels of phospho-STAT5 and Myc mRNA, suggesting that Cbl deficiency stabilizes active STAT5. c-Cbl-E3 ligase activity keeps HSC proliferative capacity in check, with loss of activity leading to increased self-renewal and abnormal proliferation in a normally quiescent adult HSC compartment[26]. Itch belongs to the HECT family of ligases (E6AP C-terminal homolog) and, similar to c-Cbl, functions as a negative regulator of HSC self-renewal and proliferation through Notch, a known regulator of hematopoiesis[26]. Fbw-7 is an E3 finger RING ligase that plays a role in the regulation of about 20 proteins including c-myc and Notch. Germline deletion of Fbw7 leads to embryonic lethality around E10.5 due to vascular defects resulting from stabilization of Notch4 in the embryo. Conditional deletion of Fbw7 leads to an increase in the frequency of active cycles of HSCs with their eventual loss. Fbw7^−/−HSC defects are cell-autonomous and include downregulation of genes involved in HSC quiescence, with an overall loss of quiescence characteristics[26].

USP1 is a DUB cysteine ​​protease that deubiquitinates FANCD2 (Faconi anemia group D2), a regulator of Faconi anemia (FA) signaling. It is able to protect HSCs from DNA damage during the divisions required to maintain the stem cell pool (see ref).[26]). RelB and nuclear factor κB (NF-κB2), major effectors of aberrant NF-κB signaling, intrinsically up-regulate HSC and renew progenitor cells[45].

4.1 Connective Tissue Growth Factor (CTGF)

CTGF is a mitogenic heparin-binding protein secreted by fibroblasts and other connective tissue cells following stimulation with TGF beta (Grotendorst, 1997;Ramazani et al., 2018). It is a member of the CCN family of regulatory proteins that modulate the ECM and share structurally related functional domains—such as cysteine-rich angiogenic inducer 61 (CYR61);overexpressed nephroblastoma protein(NOV) and WNT1-induced signaling pathway proteins WISP1, WISP2 and WISP3. CTGF is a downstream mediator of TGF-beta that stimulates connective tissue cell proliferation and ECM synthesis. It has been reported not to modulate epithelial or immune cells (Grotendorst, 1997;Ramazani et al., 2018).

Most CCN family members share a similar structure with four conserved domains: 1) an insulin-like growth factor-binding protein domain, 2) a von Willebrand factor C repeat, 3) a CCN family repeat. thrombospondin types 1 and 4) a cysteine ​​knot (Grotendorst, 1997;Ramazani et al., 2018). The hinge between domains 2 and 3 can be cleaved by various proteases. Each domain has been shown to have specific biological functions, but they also work together in crosstalk between different signaling pathways. CTGF binds structural components of the ECM important for TGF-beta signaling, including perlecan and decorin, discussed later in this review, with integral cell surface molecules such as integrins and connexins 43 (June and Lau, 2011;Aguiar et al., 2014).

Transcription of CTGF mRNA is induced by many stimuli, including growth factors and cytokines, hormones, mitogens, mechanical stress, UV light, and oxygen deprivation.Kubota and Takigawa, 2015). Most TGFβ-mediated responses involve stimulation of CTGF at some level, including stimulation of ECM component production, fibroblast proliferation, and other processes during wound healing and fibrosis.Xue et al., 2015;Cho and others, 2015), but there is often interaction between the stimuli. For example, the synergy between hypoxia and TGF-β signaling increases CTGF expression in muscle fibers but not in fibroblasts (Valle-Tenney et al., 2020). In other pathophysiological processes, CTGF appears to function independently of TGF-beta. For example, CTGF is not upregulated by normal TGF-β stimulation shortly after skeletal muscle denervation, and other factors are likely involved in fibrotic responses in these tissues.Rebolledo et al., 2019). Many miRNAs are involved in the regulation of CTGF expression.Ramazani et al., 2018).

Some attention has been paid to the potential differential effects of TGF beta-1. -2 or -3 in CTGF mRNA expression. In a mouse model study of anti-thy 1.1 glomerulonephritis (Ito et al., 2001), while differential expression of the three TGF-beta isoforms emerged over timeno partAfter injury, all three isoforms were found to be equivalent in regulating CTGF mRNA in cultured mesangial cells and glomerular epithelial cellsin vitro. In another study in which primary human fibroblasts were isolated in culture from hypertrophic scars, keloid or normal skin (Colwell et al., 2005) increased CTGF expression in normal fibroblasts more than 150-fold when stimulated with TGF beta-1, but also more than 100-fold when stimulated with TGF beta-2 or TGF beta-3 stimulated. CTGF expression was higher after TGF-beta-1 stimulation in hypertrophic circular fibroblasts compared to TGF-beta-2 and TGF-beta-3, however, there was still significant upregulation of CTGF by all three TGF -beta-isoforms. CTGF expression of keloid fibroblasts was also increased more than 100-fold after stimulation with TGF beta-1 and even more than 75-fold after addition of TGF beta-2 or TGF beta-3. Based on the hypothesis of an antifibrotic role of TGF beta-3, one would expect that TGF beta-3 would reduce CTGF expression, but this does not appear to be the case. However, this needs to be studied in other tissues before any firm conclusions can be drawn about it.

CTGF contributes significantly to renal, cardiovascular and skeletal development, with TGF-beta also heavily involved.Ramazani et al., 2018). It is also involved in the development of fibrosis in most organs, including the heart, kidney, liver, lung, skin and eyes, where TGF beta plays an important role (Ramazani et al., 2018). It is also involved in TGF-beta-related carcinogenesis and tumor growth.Wells et al., 2015). Therefore, CTGF also plays a role in many processes in which TGF-beta plays an important role.