Читать книгу Influence of FOX genes on aging and aging-associated diseases - Elena Tschumak - Страница 11

Structure of the FoxP2 gene and its isoforms

The FOXP2 gene is located on chromosome 7 and contains at least 280 (according to some data 603) kb, many introns (about 280,000 non-coding base pairs, according to a publication in 2007 - 603,000 base pairs), 7 exons ( 2145 coding base pairs), but their number is variable. (Zhang et al., 2002;Wright and Hastie, 2007)

The protein product of the FOXP2 gene consists of 715 amino acids and has the following domains: a highly conserved DNA-binding domain, a 508 to 584 amino acid "winged" helix domain (BHT) and the forkhead box with highly conserved two beta-sheets, three alpha helixes and a helix-turn-helix-motif-wing.

Structural variations occur between the second and third helix. The polyglutamine-rich regions with the repetitive CAG and CAA sequences show a high mutation rate as well as different length in different taxa. The FOXP2 gene has a zip finger involved in protein-protein interactions and a leucine zipper. The DNA binding in the minor and the major groove to various targets occurs between the third alpha helix (recognition helix) and the second wing of the FOX transcript. (Enard et al., 2002;Kaestner et al., 2000; MacDermot et al., 2005) The hinge loop plays the most important role in the FOXP2 protein binding to the target genes and the mutation P539A changes its form.(Morris et al., 2018)

Alternative splicing creates different FOXP2 isoforms and causes changes in FOXP2 activity. (Castellano and Downward, 2011). Depending on the tissue and cell type, FOXP2 expression can be started on at least 4 starting points (TSSs). (Bruce and Margolis, 2002; Schroeder and Myers, 2008).

Regulation of various genes by dimer formation with FOXP genes

FOXP2 cooperation and FOXP homo- and heterodimers A strong cooperation between FOXP members could be due to the fact that the FOXP2, FOXP1, FOXP3, and FOXP4 are 55-65% identical. A possible explanation for this similarity is proposed by Song et al. in "Genesis of the vertebrate FoxP subfamily member genesis during two ancestral whole genome duplication events" (2016). According to Murugan et al. (2013) decreased FoxP2 expression in the striatal region of adult zebra finches also interferes with their sensitivity to dopaminergic regulation in signalling via D1 receptors in Area-X, they also have co-localized dopamine D1A, D1B and D2 receptors in striatal Foxp2-expressing neurons. Dopamine is considered to be an important neurotransmitter whose deficiency causes some neurodegenerative diseases, e.g. aging relevant Parkinson's disease. This disease is characterized among other things by a washed-out language. It would be interesting to investigate if FOXP2 plays a role in these processes. The FoxP proteins can regulate their target genes in various cellular contexts depending on binding cofactor. Different protein combinations can lead to opposite effects. This could explain why certain tissues might be much more susceptible to the effects of mutations than other tissues. This study opened new perspectives in the regulation of FOXP2 target genes via protein-protein interactions between the FoxP family members, enabling a deeper understanding of the combinatorial control between the FoxP2 and its interaction partners. This study provided new basic knowledge not only about birdsong, but also about the neural function of human speech.

FOXP2 regulates the expression of many genes during embryonic development as well as the WNT and Notch signalling pathways. Further interactions have been observed with some histone family members (H2AFX; H3f3B) and two heat shock proteins (Hsp25; Hsp90a).

Importance of FOXP1/2/4 interaction for oncological processes Several FOXP genes have been observed in many aging relevant oncological processes. Foxp1 / 2/4-NuRD interaction is processed by the p66beta, a transcriptional repressor and a component of NuRD. During this process, the chromatin-remodelling complex regulates gene expression. He also influences the Interleukin-6. Interleukin-6 in turn contributes to the epithelial injury response and activates „myeloid cells“. The „myeloid cells“ are generally associated with cancer and stimulate eg. intestinal cells to divide, which leads to colorectal carcinoma. Artavanis-Tsakonas et al. (1999) studied NCOR2 and SNW1 as part of notch-mediated signalling and its role in proliferation and differentiation processes as well as in apoptosis. The NCOR2 is not only a FOXP2 downstream target but also shows interaction with FOXP1 during myocardial development. (Jepsen et al., 2008), (Wilke et al., 2012) NCOR2-mediated regulation can be considered as a common mechanism by which FOXP1 and other members of the FOXP family regulate gene expression during organogenesis. This study showed different effects of six FOXP1 / 2/4 protein combinations on the NCOR2. The FOXP1 / 2 combination showed the strongest effect. All combinations except the FOXP1 / 4 dimer led to increased NCOR2 expression. The FOXP2 homodimers induced decreased SNW1 expression while the FOXP1 and FOXP4 homodimers led to increased expression. The influence of FOXP1 / 2 and FOXP4 / 2 on SNW1 expression seems to be unlikely. These results gave an interesting insight into the dual FOXP2 function both as a repressor and as an activator. This ability for build different dimer-combinations may be a hint to fine-tune cell-specific transcriptional regulation. The FOXP1 / 2/4 dimer combinations are preferred. These results suggest that relative levels of FOXP1 / 2/4 proteins determine FOXP2's ability to act as an activator or repressor. The researchers found also that FOXP1 / 2, FOXP1 / 4 and FOXP2 / 4 are co-localized.

FOXP2 modulation by alternative splicing The FOXP is also regulated by alternative splicing. This way the FoxP2 gets different isoforms and is homodimerized and this leads to a change of its activity. (Santos et al., 2011) Similar results were reported by Chen et al. (2014) and Olias et al. (2013). This FOXP2 modification was observed in the lower and dorsal thalamus, in the striatum (except Area X) and in the cerebellar Purkinje cells. These are brain areas where the FoxP2 is permanent strongly expressed. (Takahashi et al., 2003), (Ferland et al., 2003), (Haesler et al., 2004) Epigenetic FOXP2 Regulation by Methylation FOXP2 methylation plays an important role by adipositas. Spaeth et al. showed in „The FOXP1, FOXP2 and FOXP4 transcription factors are required for islet alpha cell proliferation and function in mice“ (2015) that Foxp2 is important for the growth and function of pancreatic alfa islands. The islets of Langerhans of the exocrine pancreas contain five different cell populations: the beta, the alpha, the delta, the epsilon and the pancreatic polypeptide cells. These secrete the ghrelin and pancreatic polypeptide hormones (insulin, glucagon, somatostatin). The authors noted that interaction between FOXP2 and FOXP1 / FOXP4 is required for alpha-islet cell proliferation of the mice. Adult beta cells normally secrete insulin, the alpha cells - the glucagon. Autoimmune beta-cell destruction causes type 1 diabetes while type 2 diabetes is characterized by insulin resistance in the peripheral tissues. Type 2 diabetes is accompanied by insulin deficiency and the loss of beta cell identity. The transcription factors accomplish the reprogramming of terminally differentiated cells and embryonic stem cells into the beta-like cells. The members of the FOX superfamily play crucial roles in these processes, which is also aging relevant. E.g., FOXA2 is known as a pancreatic cell fate regulator. FOXM1 controls expression of cycle factors and increases beta cell mass during metabolic stress, including pregnancy stress and partial pancreatectomy. Metformin, Berberine, EGCG, quercetin and other natural products activate cancer relevant AMPK and decrease mTORC1 activity. MTORC1 activity is frequently elevated in CSC including pancreatic CSCs (Ming et al., 2014; Rozengurt et al., 2014; Matsubara et al., 2013) EGCG also positively effects cancer and aging relevant p21Cip1 and negatively the PI3K/PTEN/Akt/mTORC1 pathway (Chen et al., 2012)

According to Alessandro et al., (2015) MET positively influences decreased mitochondrial transmembrane potential, sensitivity to TRAIL via DR5-upregulation, ROS-level and cell cycle in cancer via miR-221 (Tanaka et al., 2015 Matsubara et al., 2013) but also via TRAIL interaction. In this case G-Phase arrest happens via p27Kip-1 and via interaction between caspases (Coleman et al., 2013) Other plant-derived cancer relevant chemicals are e.g. SHH pathway regulating catechins cyclopamine, norcantharidin, sulforaphane and zerumbone (Huang et al., 2013) They act best in combination and suppress ALDH1, MMP-2 and MMP-9 via KRAS under- and let7 miR- upregulation. (Appari et al., 2014) Momordica charantia has positive effect on inflammation and on cancer (Dandawate et al.,2016), e.g. on ovarian cancer via of AMPK up-, mTOR/p70S6K and AKT/ERK/FOXM1 signalling cascade underregulation (Yung et al., 2016) possibly via Alpha-Momorcharin (Deng et al., 2014).

Also Rooperol influences apoptosis with the help of mitochondrial membrane potential. It upregulates ROS via TP53 activation, but also OCT4 and stemness relevant SOX2 and NANOG (Ali et al., 2015) It also increases Pomiferin level, which influences BIM1 and NESTIN (Zhao et al., 2013)

FOXP1, FOXP2 and FOXP4 are of great importance for alpha cell proliferation and function and are expressed in the pancreas and eyes of Xenopus laevis during its development. FOXA1 and FOXA2 also regulate glucagon production and secretion-controlling genes: the MAFB, the Brn4 (also known as the Pou3f4), the PCK2, the Nkx2-2, the Kir6.1 (also known as the Kcnj8), the Sur1 (also known as the ABCC8) and the GIPR.

FOXP2 regulation by external factors Regulation by PH level Blane and Fanucchi (2015) studied in "Effect of pH on the Structure and DNA Binding of the FOXP2 Forkhead Domain." effects of pH from changes 5 to 9 on FOXP2 function and reported that a change in pH (pH 7.5) directly affects the FOXP2 binding affinity via the altered hydrogen bonding pattern. This is due to the protonation or deprotonation of His554 (the amino group of its imidazole side chain, pKa ~ 6.5). The researchers used as methods gel permeation chromatography, ultraviolet circular dichroism, intrinsic and extrinsic fluorescence etc. Their results showed that the pH does not affect the protein secondary structure in the presence or absence of DNA but alters its tertiary structure. The protein showed a less compact structure at low pH in the absence of DNA. When the DNA was added, the protein became more compact, even at low pH and its dimerization potential increased. They regarded the pH as a regulatory mechanism of FOXP2 forkhead domain (FHD) transcription that interacts with the DNA by helix placement in the major groove. These results could also be important in cancer tissue, where FOXP2 expression plays an important role. E.g., not only the genetic component but also a previous chronic gastric ulcer with changed pH plays a significant role in gastric cancer. The gastric ulcer-causing Helicobacter pylori gains an almost pH-neutral environment with the help of the enzyme urease, which splits the urea into carbon dioxide and ammonia. It would be of great scientific interest to investigate whether the change in the gastric pH together with FOXP2 have an effect on the development of gastric cancer.

Regulation through Vitamin-D

Hawes et al. (2015) showed in „Maternal vitamin D deficiency age foetal brain development in the BALB / c mouse“ how the maternal vitamin D deficiency alters foetal brain development in transgenic Balb / c mice. Before and during pregnancy a vitamin D-rich (2.195 IU / kg) or a low (0 IU / kg) diet was given for 5 weeks and the foetal brains were analysed morphologically and for gene expression at 14.5 or 17.5. embryonic day. It was found that the vitamin D deficiency during pregnancy leads to reduction of rump length, lateral ventricle volume and head size. The FoxP2 expression and at the same time the expression of Brain-Derived Neurotrophic Factor (BDNF), the Transforming growth factor-β1 (TGF-β1) and brain tyrosine hydroxylase (TH) in dopaminergic neurons was altered. The vitamin D-poor diet reduced FOXP2 expression in immunoreactive cells and in the developing cortex in female foetuses. These results allowed deeper insight into the medically relevant reasons for foetal degeneration accompanied by prenatal vitamin D deficiency. It is known that vitamin D interacts with neurotrophic factors (NGF, NT3, NT4, GDNF) and its deficiency triggers neurodegenerative diseases in old age. (Jia et al, 2015) It would be of great interest to investigate if this effect is based on the interaction with FOXP2. Since FOXP2 seems to play an important role in many oncological diseases it would be also interesting to investigate if and to what extent this influence is controlled by vitamin D.

FOXP2 regulation by internal factors Regulation by HuR According to Popovitchenko et al. (2016) „Depending on its degree of phosphorylation, the antigen R (HuR) dictates the amount of FOXP2 mRNA and the development of the neocorticals controlled by it, depending on its degree of phosphorylation“ HuR is the main factor in differential translation of autism-associated FoxP subfamily members in the developing neocortex subpopulation of the projection neurons. Regulation by Risperidone and NAP The study of a big Scottish family with severe mental disorders and schizophrenia (Liu et al. 2009) showed a balanced chromosomal translocation [(1:11) (q42.1; q14.3)] and an abnormally-truncated DisC1 protein (a microtubule-regulating and the FOXP2-influenced protein encoded on chromosome 1). The DISC1 is expressed in the cerebral cortex, in the hypothalamus, in the amygdala, in the hippocampal dentate gyrus, in the olfactory bulb and in the cerebellum. The truncated human DISC1 (hDISC1) alters its localization and can no longer interact with microtubules and microtubule-associated proteins. It leads to decreased dendritic growth and branching. Such anomalies have also been found in brain samples from patients with schizophrenia. (Harrison, 2004) This mutation has also been linked to depression and bipolar disorder. (Burdick et al., 2005) A connection between DISC1 and FOXP2 effects human verbal fluency as well as the ability to acquire spoken language. Several studies indicate that FOXP2 polymorphisms are in some cases associated with schizophrenia. (Nicodemus et al., 2014), (Walker et al., 2012), (Lai et al., 2001), (MacDermo et al., 2005), (Sanjuan et al., 2005), (Sanjuan et al. , 2006), (Tolosa et al., 2010) Vaisburd et al. (2015) showed in „Risperidone and NAP protect cognition and normalize gene expression in a schizophrenia mouse model“ that risperidone reduces effects of the DISC1 mutation. Risperidone is an analogue of the microtubule-stabilizing activity-dependent neuroprotective protein (ADNP) with a NAP (NAPVSIPQ) sequence and a SxIP motif (a microtubule junction for microtubule-end binding, responsible for microtubule dynamics and this is also important for synaptic plasticity and neuroprotection). (Oz et al., 2014), (Gozes et al., 2011), (Holtser-Cochav et al., 2006), (Jouroukhin et al., 2013), (Kumar & Wittmann, 2012).

Both risperidone and NAP improved object recognition in the Morris water labyrinth. In contrast to Risperidone, NAP additionally reduced the anxiety in transgenic mice. Doxycycline blocked the expression of the mutated DISC1 gene. The candidate drugs were selected using bioinformatics programs and then affinity chromatographed. Mutations of the DISC1 gene were associated with increased FOXP2 level in hippocampus. FOXP2 levels could be significantly reduced by treatment with NAP, risperidone or their combination. This effect may be due to the blocking of the dopamine and 5HC2A serotonin receptors in the mesolimbic system, which leads to the reduction of negative schizophrenia symptoms. (Meltzer and McGurk, 1999), (Farde et al., 1995)

An increased dopamine level leads to psychotic symptoms. The study by Mendoza et al. (2015), showed that FoxP-expressing neurons in Area-X also contain dopamine receptors 1A, 1B, and 2. Further studies may clarify whether the schizophrenic positive symptoms are due to the dopamine-FOXP2 interaction and how much FOXP 2 can influence dopamine dependent neurodegeneration in aging.

Regulation of FOXP2 and other FOXP genes by SUMOilization

Effect of sumoilization on the FOXP2 gene In „The Key Regulator for Language and Speech Development, FOXP2, is a Novel Substrate for SUMOylation“ Meredith et al. (2016) reported that the FOXP2 is covalently modified on its amino acid residues by both SUMO1 and SUMO3 at phylogenetically conserved lysine 674. The acid residues downstream of the FOXP2 SUMOylation motif are required for full SUMOylation capacity and modulation of its downstream target genes (DISC1, SRPX2 and MiR200c). Sumoylation is a form of posttranslational modification. The SUMOs (small ubiquitin-like modifiers) is a class of regulatory proteins that control the activity of the target protein and its interaction with other proteins. But the modification does not alter the localization and stability of FOXP2. The modulation effect can be reduced by SENP2 (a specific SUMOylation protease). It has also been observed that the human R553H mutation of FOXP2 reduces its SUMOylating ability compared to wild-type FOXP2. Isui et al. (2017) showed in „Sumoylation of FOXP2 Regulated Motor Function and Vocal Communication Through Purkinje Cell“ with the help of in utero electroporation that the SUMOylation of FOXP2 has, among others, an effect on the development of Purkinje cells and plays an important role in motor skills and vocal communication. They identified FOXP2 SUMOylation in the cerebellum of new-born K673 mice with the help of in vivo and in vitro coimmunoprecipitation and concluded that PIAS3 (an E3 ligase of the small ubiquitin-like modifier) catalyses FOXP2 SUMOylation. This SUMOylation modifies the transcription regulation of FOXP2. Estruch et al. (2016) showed in „The language-related transcription factor FOXP2 is post-translationally modified with small ubiquitin-li modifiers“ that post-translational FOXP2 modification is accomplished by members of the PIAS family. These members of the PIAS family are E3 ligases that promote SUMO transfer from the SUMO-conjugating enzyme UBC9 to an acceptor lysine residue of the target protein. (Rytinki et al., 2009) The SUMO2 and the SUMO3 are 95% identical. However, SUMO1 is only to 50% identical to SUMO2 and SUMO3 (Meulmeester and Melchior, 2008). Estruch research group studied various DNA constructs. They used myc-labelled PIAS1 and the His, mCherry and the YFP-labelled SUMOs and the bioluminescence resonance energy transfer assays. For FOXP2 detection they used Western blotting with the anti-V5 antibody and the anti-beta actin as reference. They took UBC9 enzyme to measure the extent of FOXP2 SUMOylation. During the study of FOXP2-PIAS interaction the working group used the BRET assay and the luciferase assays with the Renilla luciferase (Luc). Further methods were the gel-shift assay, the pull-down assay, the yeast two-hybrid assay and the fluorescence microscopy of HEK293 cells. The study revealed that FOXP-2 could be modified by all three human SUMO proteins and that PIAS1 promotes this process. The docking site of SUMO proteins in the FOXP1 and FOXP4 appears to be the most conserved N-terminal region of FOXP2 away from the polyglutamine tract. This region also interacts with the autism-relevant transcription factor TBR1. (Parikshak et al., 2013)

Biochemical aspects of FOXP2 SUMOylation C-terminal carboxyl groups of SUMOS appear to interact with the amino group of FOXP2 via an isopeptide linkage. This amino group of the FOXP2 protein has a lysine side chain with the ΨKX (where Ψ is a hydrophobic amino acid and X is any other amino acid) consensus sequence . The SP-RING domain of the SUMO helps its FOXP2 recognition. N-terminal FOXP2 region contain some of the most important determinants of PIAS binding. These are K674 with PE motif and other residues, e.g. K285, K417 and K560 of the VE sequence. All of these residues are conserved in FOXP2. In this experiment a for SUMO conjugation required Arginine was removed and the SUMOLlyation did not happened. This showed that the K674 is the most important determinant for the SUMO1, SUMO2 and SUMO3 modifications. The mutants assay showed normal dimerization ability in the BRET, so the researchers concluded that the suppressed SUMOylation does not affect the FOXP2 ability to form homodimers or to interact with the CtBP129 co-repressor. This is because FOXP2 dimerization occurs with the help of the leucine zipper domain. However, the leucine zipper domain is not located near the SUMOylation site. This region includes domains that are highly conserved in FOXP1 and FOXP4. These domains serve as recognition sites for the autism-relevant transcription factor TBR1. This area is probably responsible for several protein-protein interactions. (Deriziotis et al., 2014) SUMOilization in FOXP1 / 4 genes Three SUMO proteomic studies of human cell lines identified FOXP1 and FOXP4 as substrates for SUMOilination. (Golebiowski et al., 2009; Tatham et al., 2011; Wen et al., 2014) FOXP1 and FOXP4 contain the SUMOylation relevant DNA motif that is identical to the corresponding FOXP2 motif of the surrounding PE sequence. In BRET study, FOXP1 also showed interactions with PIAS1, PIAS2, and PIAS4, but in contrast to FOXP2, FOXP1 showed little or no interaction with PIAS3. The FOXP1 also showed interactions with all three SUMOs. Probably FOXP1 is also Sumoylated by SUMO1, SUMO2 and SUMO3 at a location, like the K636 site of FOXP2. So, members of the PIAS family are act different in FOXP1 and FOXP2 SUMOylation.

Influence of FOXP2 SUMOlyatin on various diseases It is known that not only FOXP1 and FOXP4 transcription factor TBR1 and co-repressor CtBP1 interact with FOXP2. (Deriziotis et al., 2014; Li et al., 2004; Meulmeester and Melchior, 2008; Rytinki et al., 2009). So by speech and language disorders afflicted family showed a strongly reduced FOXP2 SUMOylation. SUMOylation appears to occur in thousands of nuclear proteins in all cell types at different stages of development (Meulmeester and Melchior, 2008; Gwizdek et al., 2013). According to Bacon and Rappold (2012) and Zhao et al. (2014) SUMO influences dendritic and synaptic morphogenesis by modifying neuronal proteins. Different PIASs may be involved in SUMOylation of various FOXPs and may affect the modification of neuronal circuits. In addition, they support the neuronal plasticity, but it is unclear whether a change in SUMOylation level during brain development leads to functional impairment. It has been demonstrated that PIAS1 knockout causes perinatal lethality in mice. (Loriol et al., 2012; Hasegawa et al., 2014) The study of Estruch et al. (2016) revealed that the four PIAS were continuously expressed but the PIAS1 and PIAS3 were expressed at the same extent in all tissues tested while the PIAS2 and PIAS4 were expressed primarily in testis. Studies of Turner et al. (2013), Adegbola et al. (2015), Nazaryan et al. (2014), Utine et al. (2014) and the fact that the FOXP2 plays a role in neuronal migration, neurite growth, in synaptic plasticity and in the language-related brain circuits (Garcia-Caleroet al., 2015; Tsui et al., 2013; Verne et al., 2011; Groszer et al., 2008) allowed another hypothesis about the cause of speech disorders in the KE family. All members of the by the speech disorder afflicted KE family had a heterozygous R553H mutation in the DNA-binding domain. In twenty other similar cases a missense mutation (R553H) in the FOX DNA binding domain that makes DNA binding and transcriptional repression by FOXP2 impossible was found. Nonsense and frameshift mutations, as well as chromosomal rearrangements were accompanied by similar disabilities. In contrast to the partially compensated interactions between the SUMOS and the K674R mutant the BRET assay showed an almost complete loss of the interactions between the SUMOS and R553H mutant compared to the wild type. The most important SUMOylation site stayed intact in the R553H mutants so the reduction of SUMOylation may base on reduced or absent interaction between the mutant and the PIASs or UBC9. In contrast to the K674R mutants which interact with PIAS but cannot be SUMOylated, the R553H mutants had reduced interaction with PIAS, which can be compensated by PIAS overexpression or by a fusion with UBC9. The R553H mutation also led to partial mislocalization and increased protein aggregation. The R553H mutation may lead to a conformational change that blocks the PIAS binding site. Alternatively, the loss of DNA binding capacity resulting from the R553H mutation and / or destabilization of the protein domain may trigger FOX interactions with other cellular proteins that interfere with PIAS binding. It seems to be useful to investigate the extent of the FOXP2 SUMOylation and the FOXP2-PIAS interactions in different issues and possibly further regulatory mechanisms of aging related neurodegeneration.

FOXP2 regulates proto-oncogenes p21WAF1 / RAS 1, BCL-2, HES1 and other cancer-relevant genes. Gascoyne et al. (2015) demonstrated in „The forkhead transcription factor FOXP2 is required for regulation of p21WAF1 / CIP1 in 143B osteosarcoma cell growth arrest“ with the help of human osteosarcoma cell cultures and normal human osteoblasts that the forkhead transcription factor FOXP2 regulates proto-oncogenes p21WAF1 and CIP1. These proto-oncogenes are required for osteosarcoma cell growth arrest. lncRNA-p21 influences cell proliferation and carcinogenesis via vβ-catenin and JunB. The lncRNA HULC is highly expressed in age related colorectal carcinomas and UCA1 is highly expressed in bladder transitional cell carcinoma by modulating p27 level and can affect cellular senescence.(Parket al., 2011; Smith-Vikos and Slack, 2012) The decrease of growth factor level as well as MAPK reduction induces FOXP2 expression. In turn FOXP2 expression triggers cyclin-dependent kinase inhibitor p21WAF1 / CIP1 expression. It is known that p21 is one of the p53 target genes. (Deiry et al., 1994)19ARF/p53 and p16INK4a/Rb stop cancer cells from proliferation and play an important role in cellular senescence.

During these experiments, the DMEM-cultured and ATCC-derived osteosarcoma cell lines were treated with the IKK pathway inhibitor VII, the MAPK pathway inhibitor, the LY-294002 PI3K pathway inhibitor, the 117082 NF-kappa B pathway inhibitor, the Notch signalling pathway inhibitor or with the DMSO vesicle. Then the alkaline phosphatase and MTS assays were done and 405nm absorbance was measured. Other methods were RNA isolation, cDNA preparation and real-time PCR, protein isolation, immunoblotting, chromatin immunoprecipitation and flow cytometric analysis of the cell cycle distribution. The authors found a link between the 7-time increased FOXP2 expression in murine bone 48 h after treatment and the decreased growth of osteosarcoma cell lines. Unlike mesenchymal tissue osteoblasts did not require RUN involvement in FOXP2 induction. (Zhao et al., 2014) In neuroblastoma cells growth arrest was not accompanied by FOXP2 induction. Cyclin-dependent kinase inhibitors such as p21WAF1, p27KIP1, CIP1CDKN1A and CDKN1B organize CDK inhibition. These experiments also demonstrated that FOXP2 induces CDKN1A / p21 expression in osteoblasts and serves as a primary trigger for cell growth arrest. FOXP2 reduction resulted in strong CDKN1A / p21 and slight CDK4 decline and showed no effect on p53 level. Increased p53 / p21 level had no effect on FOXP-2 level. Immunoprecipitation did not show direct FOXP2 / p21 binding sites and it was demonstrated that although increased FOXP2 expression was accompanied by concomitant IL-6 expression but FOXP2 did not controls p21 expression via IL-6, p21 induction by the FOXP2 takes place indirectly. This is still to be studied. The researchers found a correlation between Lhx8 deficiency factor and FOXP2 expression. There may be a relationship between the FOXP2 and the Soxp2.

Yan X et al. showed 2015 in „Downregulation of FOXP2 promoter human hepatocellular carcinoma cell invasion“ that FOXP2 level in human hepatocellular carcinoma cells is significantly reduced compared to adjacent non-tumor tissue. They used Western blot, transwell assays and immunohistochemistry to measure FOXP2 expression in HCC and in adjacent normal tissues of 50 patients. Their results showed that FOXP2 expression was down-regulated in HCC tumor tissue and that FOXP2-level decrease correlated with poor survival rates. FOXP2 could decrease cell invasion and affect the expression of vimentin and OXE-cadherin.

Since the study by Schroeder and Myers (2008) has emphasized the importance of cell-specific FoxP expression it is especially important to measure it in different tissues and to take a closer look at its cell-specific interaction mechanisms. Chiu et al. (2014) described how FOXP2 inhibits the ERK / MAPK (HSPB7), NOS1, KCNJ15 and SHH pathways (PTCH1) and Vernes et al. (2011) and Ayub et al. (2013) how it influences the oncogene CDK8. FOXP2 effects Bcl-2 expression which seems to be involved in pro-caspase activation and apoptosis. (Devanna et al., 2014). Spiteri et al. (2007) described the cancer-related actin-binding protein genes TAGLN, NOS1, LBR, KCNJ15 and ANK1 as additional FOXP2 target genes. FP2 affects the proto-oncogene C-MET by binding its regulator the AP1-NFAT complex. It also affects the p53 which in turn binds to C-MMET. (Mukamel et al., 2011) (Ho and Crabtree, 2006). MAPK and TGF-β pathways are modified by lncPINT, which is also affected by the with PRC2 interacting p53.

FOXP1 functions, parallels to FOXP2 Like FOXP2's, FOXP1 plays an important role in brain development, because its haploinsufficiency is associated with speech deficits and autism. (Tamura et al., 2003; Le Fevre et al., 2013; Horn et al., 2010; Hamdan et al., 2010; Bacon and Rappold, 2011; Lien et al., 2001; Pariani et al, 2009; Chien et al., 2013; Palmerbo et al., 2013; Carr et al, 2010; Vargha-Khadem et al, 1998; Watkins et al. Brain, 2002; Reimers-Kipping et al. al., 2011; Scharff et Petri, 2011; Li et al., 2015; Song et al., 2015) However, according to „Equivalent missense variant in the FOXP2 and FOXP1 transcription factors causes distinct neurodevelopmental disorders“ (2017) of Sollis et al. the R514H mutation of the FOXP1 gene has a wider and heavier disease manifestation than corresponding p.R553H mutation of the FOXP2 gene. Other functions of the FOXP1 gene were described by Rousso et al. (2016), Wang et al. (2003, 2004), Xing et al.(2017) and Taskapilioglu et al. (2016). According to the recent study by Fröhlich et al. „Foxp1 expression is essential for sex-specific murine neonatal ultrasonic vocalization“ (2017), Foxp1 expression is essential for sex-specific murine neonatal ultrasound vocalization. This study could be of clinical importance because boys are more affected by utism and other speech and speech deficits than girls. They showed that Foxp1 and the androgen receptor are co-expressed in striatal neurons and that brain-specific androgen receptor KO (ArNesCre) mice have a reduced Foxp1 expression in the striatum and an increased Foxp2 level. Oestrogen also reduced the FOXP1 level in breast cancer tissue. (Shigekawa et al., 2011) FOXP3 significance for Alzheimer's disease and for apoptosis Baruch et al. (2015) highlighted the importance of FOXP3 in Alzheimer's disease, in with chronic inflammation leads to the development of myeloid plaques. This inflammation is caused by a loss of Foxp3 + regulatory T cells (Tregs). It would be interesting to investigate whether FOXP3 modulation plays a role in Alzheimer's disease, as it is the case with FOXO1 and FOXO3a. So FOX-miRNA modulation is involved in the progression of Alzheimer's disease. (Lau et al., 2013). This study „Alteration of the microRNA network during the progression of Alzheimer's disease“ observed the hippocampal miRNAs dysregulation of AD-patients. In them miR-132-3p expression on chromosome 17 was altered in both hemispheres. In AD stages III and IV it was severely decreased (41 persons with late-stage AD were compared to 23 healthy volunteers). This dysregulation was mainly observed in tau hyperphosphorylated neurons. This miRNA may affect both the TAU and the transcription factor FOXO1, whose expression is increased in the LOAD hippocampus. According to Wong et al. (2013), miR-132-3p also regulates FOXO3a, which in turn activates pro-apoptotic genes and is overexpressed in LOAD