S100 Proteins-Intracellular and Extracellular Function in Norm and Pathology.

The S100 proteins are small, ubiquitous, mostly homodimeric proteins containing two EF-hand structures, that is, helix-loop-helix motifs specialized in high-affinity calcium-binding (~10-6 M) [...].

Dynamics and Epigenetics of the Epidermal Differentiation Complex.

Epidermis is the outer skin layer built of specialized cells called keratinocytes. Keratinocytes undergo a unique differentiation process, also known as cornification, during which their gene expression pattern, morphology and other properties change remarkably to the effect that the terminally differentiated, cornified cells can form a physical barrier, which separates the underlying tissues from the environment. Many genes encoding proteins that are important for epidermal barrier formation are located in a gene cluster called epidermal differentiation complex (EDC). Recent data provided valuable information on the dynamics of the EDC locus and the network of interactions between EDC gene promoters, enhancers and other regions, during keratinocytes differentiation. These data, together with results concerning changes in epigenetic modifications, provide a valuable insight into the mode of regulation of EDC gene expression.

Involvement of CacyBP/SIP in differentiation and the immune response of HaCaT keratinocytes.

CacyBP/SIP is a multifunctional protein present in various cells and tissues. However, its expression and role in the epidermis has not been explored so far. In this work, using RT-qPCR, Western blot analysis and three-dimensional (3D) organotypic cultures of HaCaT keratinocytes we show that CacyBP/SIP is present in the epidermis. To investigate the possible role of CacyBP/SIP in keratinocytes we obtained CacyBP/SIP knockdown cells and studied the effect of CacyBP/SIP deficiency on their differentiation and response to viral infection. We found that CacyBP/SIP knockdown results in reduced expression of epidermal differentiation markers in both undifferentiated and differentiated HaCaT cells. Since epidermis is engaged in immune defense, the impact of CacyBP/SIP knockdown on this process was also analyzed. By applying RT-qPCR and Western blot it was found that poly(I:C), a synthetic analog of double-stranded RNA that mimics viral infection, stimulated the expression of genes involved in antiviral response, such as IFIT1, IFIT2 and OASL. Interestingly, following poly(I:C) stimulation, the level of expression of these genes was significantly lower in cells with CacyBP/SIP knockdown than control ones. Since the signaling pathway mediating cellular responses to viral infection involves, among others, the STAT1 transcription factor, we measured its activity using luciferase assay and found that it was lower in CacyBP/SIP knockdown HaCaT cells. Altogether, the presented results indicate that CacyBP/SIP promotes epidermal differentiation and might be involved in response of the skin cells to viral infection.

S100A6 Protein-Expression and Function in Norm and Pathology.

S100A6, also known as calcyclin, is a calcium-binding protein belonging to the S100 protein family. It was first identified and purified more than 30 years ago. Initial structural studies, focused mostly on the mode and affinity of Ca2+ binding and resolution of the resultant conformational changes, were soon complemented by research on its expression, localization and identification of binding partners. With time, the use of biophysical methods helped to resolve the structure and versatility of S100A6 complexes with some of its ligands. Meanwhile, it became clear that S100A6 expression was altered in various pathological states and correlated with the stage/progression of many diseases, including cancers, indicative of its important, and possibly causative, role in some of these diseases. This, in turn, prompted researchers to look for the mechanism of S100A6 action and to identify the intermediary signaling pathways and effectors. After all these years, our knowledge on various aspects of S100A6 biology is robust but still incomplete. The list of S100A6 ligands is growing all the time, as is our understanding of the physiological importance of these interactions. The present review summarizes available data concerning S100A6 expression/localization, interaction with intracellular and extracellular targets, involvement in Ca2+-dependent cellular processes and association with various pathologies.

S100A6 as a Constituent and Potential Marker of Adult and Cancer Stem Cells.

Adult or tissue stem cells are present in various tissues of the organism where they reside in a specific environment called the niche. Owing to their ability to generate a progeny that can proliferate and differentiate into specialized cell types, adult stem cells constitute a source of new cells necessary for tissue maintenance and/or regeneration. Under normal conditions they divide with a frequency matching the pace of tissue renewal but, following tissue damage, they can migrate to the site of injury and expand/divide intensively to facilitate tissue repair. For this reason much hope is being placed on the use of adult stem cells in regenerative therapies, including tissue engineering. Identification and characterization of tissue stem cells has been a laborious process due to their scarcity and lack of universal markers. Nonetheless, recent studies, employing various types of transcriptomic analyses, revealed some common trends in gene expression pattern among stem cells derived from different tissues, suggesting the importance of certain genes/proteins for the unique properties of these cells. S100A6, a small calcium binding protein, has been recognized as an important factor influencing cell proliferation and differentiation. Accumulating results show that S100A6 is a constituent of adult stem cells and, in some cases, may even be considered as their marker. Thus, in this review we summarize literature data concerning the presence of S100A6 in adult and cancer stem cells and speculate on its potential role and usefulness as a marker of these cells.

RNA-Seq Transcriptome Analysis of Differentiated Human Oligodendrocytic MO3.13 Cells Shows Upregulation of Genes Involved in Myogenesis.

In this work, we examined the differentiation of oligodendrocytic MO3.13 cells and changes in their gene expression after treatment with phorbol 12-myristate 13-acetate, PMA, or with RNA polymerase I (Pol I) inhibitor, CX-5461. We found that MO3.13 cells changed their morphology when treated with both agents. Interestingly, CX-5461, but not PMA, induced noticeable changes in the integrity of the nucleoli. Then, we analyzed the p53 transcriptional activity in MO3.13 cells and found that it was increased in both cell populations, but particularly in cells treated with PMA. Interestingly, this high p53 transcriptional activity in PMA-treated cells coincided with a lower level of an unmodified (non-phosphorylated) form of this protein. Since morphological changes in MO3.13 cells after PMA and CX-5461 treatment were evident, suggesting that cells were induced to differentiate, we performed RNA-seq analysis of PMA-treated cells, to reveal the direction of alterations in gene expression. The analysis showed that the largest group of upregulated genes consisted of those involved in myogenesis and K-RAS signaling, rather than those associated with oligodendrocyte lineage progression.

Epigenetic Regulation of Epidermal Differentiation.

The epidermis is the outer part of the skin that protects the organism from dehydration and shields from external insults. Epidermal cells, called keratinocytes, undergo a series of morphological and metabolic changes that allow them to establish the biochemical and structural elements of an effective epidermal barrier. This process, known as epidermal differentiation, is critical for the maintenance of the epidermis under physiological conditions and also under stress or in various skin pathologies. Epidermal differentiation relies on a highly coordinated program of gene expression. Epigenetic mechanisms, which commonly include DNA methylation, covalent histone modifications, and microRNA (miRNA) activity, modulate various stages of gene expression by altering chromatin accessibility and mRNA stability. Their involvement in epidermal differentiation is a matter of intensive studies, and the results obtained thus far show a complex network of epigenetic factors, acting together with transcriptional regulators, to maintain epidermal homeostasis and counteract adverse effects of environmental stressors.

Sgt1 Regulates α-Synuclein Subcellular Localization and Expression of Parkinson's Disease Related Genes, PINK1 and PARK9.

The SGT1 protein is highly expressed in the mammalian brain, particularly in neurons of the hippocampus and cortex, and in Purkinje cells of the cerebellum. There are literature data indicating that the protein may be involved in pathogenesis of neurodegenerative disorders such as Parkinson's disease (PD). In the present work we have found that SGT1 protected cells from the toxicity of rotenone, an agent that evokes behavioral and histopathological symptoms of PD. To gain more insight into the possible mechanism underlying the protective action of SGT1 we looked at α-synuclein subcellular distribution in HEK293 cells with an altered SGT1 level. By immunofluorescent staining we have found that in HEK293 cells overexpressing SGT1 α-synuclein was mainly localized in the cytoplasm while in control cells it was present in the nucleus. Accordingly, when SGT1 expression was silenced, α-synuclein was predominantly present in the nucleus. These results were then confirmed by subcellular fractionation and Western blot analysis. Moreover, we have found that altered level of SGT1 in HEK293 cells influenced the expression of PD related genes, PINK1 and PARK9. Altogether, our results point to SGT1 as an important factor that might be involved in the pathogenesis of Parkinson's disease (PD).

Regulation of S100A10 Gene Expression.

S100A10, a member of the S100 family of Ca2+-binding proteins, is a widely distributed protein involved in many cellular and extracellular processes. The best recognized role of S100A10 is the regulation, via interaction with annexin A2, of plasminogen conversion to plasmin. Plasmin, together with other proteases, induces degradation of the extracellular matrix (ECM), which is an important step in tumor progression. Additionally, S100A10 interacts with 5-hydroxytryptamine 1B (5-HT1B) receptor, which influences neurotransmitter binding and, through that, depressive symptoms. Taking this into account, it is evident that S100A10 expression in the cell should be under strict control. In this work, we summarize available literature data concerning the physiological stimuli and transcription factors that influence S100A10 expression. We also present our original results showing for the first time regulation of S100A10 expression by grainyhead-like 2 transcription factor (GRHL2). By applying in silico analysis, we have found two highly conserved GRHL2 binding sites in the 1st intron of the gene encoding S100A10 protein. Using chromatin immunoprecipitation (ChIP) and luciferase assays, we have shown that GRHL2 directly binds to these sites and that this DNA region can affect transcription of S100A10.

Calmodulin and Its Binding Proteins in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder that manifests with rest tremor, muscle rigidity and movement disturbances. At the microscopic level it is characterized by formation of specific intraneuronal inclusions, called Lewy bodies (LBs), and by a progressive loss of dopaminergic neurons in the striatum and substantia nigra. All living cells, among them neurons, rely on Ca2+ as a universal carrier of extracellular and intracellular signals that can initiate and control various cellular processes. Disturbances in Ca2+ homeostasis and dysfunction of Ca2+ signaling pathways may have serious consequences on cells and even result in cell death. Dopaminergic neurons are particularly sensitive to any changes in intracellular Ca2+ level. The best known and studied Ca2+ sensor in eukaryotic cells is calmodulin. Calmodulin binds Ca2+ with high affinity and regulates the activity of a plethora of proteins. In the brain, calmodulin and its binding proteins play a crucial role in regulation of the activity of synaptic proteins and in the maintenance of neuronal plasticity. Thus, any changes in activity of these proteins might be linked to the development and progression of neurodegenerative disorders including PD. This review aims to summarize published results regarding the role of calmodulin and its binding proteins in pathology and pathogenesis of PD.

Effect of SUV39H1 Histone Methyltransferase Knockout on Expression of Differentiation-Associated Genes in HaCaT Keratinocytes.

Keratinocytes undergo a complex differentiation process, coupled with extensive changes in gene expression through which they acquire distinctive features indispensable for cells that form the external body barrier-epidermis. Disturbed epidermal differentiation gives rise to multiple skin diseases. The involvement of epigenetic factors, such as DNA methylation or histone modifications, in the regulation of epidermal gene expression and differentiation has not been fully recognized yet. In this work we performed a CRISPR/Cas9-mediated knockout of SUV39H1, a gene-encoding H3K9 histone methyltransferase, in HaCaT cells that originate from spontaneously immortalized human keratinocytes and examined changes in the expression of selected differentiation-specific genes located in the epidermal differentiation complex (EDC) and other genomic locations by RT-qPCR. The studied genes revealed a diverse differentiation state-dependent or -independent response to a lower level of H3K9 methylation. We also show, by means of chromatin immunoprecipitation, that the expression of genes in the LCE1 subcluster of EDC was regulated by the extent of trimethylation of lysine 9 in histone H3 bound to their promoters. Changes in gene expression were accompanied by changes in HaCaT cell morphology and adhesion.

S100A6 and Its Brain Ligands in Neurodegenerative Disorders.

The S100A6 protein is present in different mammalian cells and tissues including the brain. It binds Ca2+ and Zn2+ and interacts with many target proteins/ligands. The best characterized ligands of S100A6, expressed at high level in the brain, include CacyBP/SIP and Sgt1. Research concerning the functional role of S100A6 and these two ligands indicates that they are involved in various signaling pathways that regulate cell proliferation, differentiation, cytoskeletal organization, and others. In this review, we focused on the expression/localization of these proteins in the brain and on their possible role in neurodegenerative diseases. Published results demonstrate that S100A6, CacyBP/SIP, and Sgt1 are expressed in various brain structures and in the spinal cord and can be found in different cell types including neurons and astrocytes. When it comes to their possible involvement in nervous system pathology, it is evident that their expression/level and/or subcellular localization is changed when compared to normal conditions. Among diseases in which such changes have been observed are Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), epileptogenesis, Parkinson's disease (PD), Huntington's disease (HD), and others.

The Effect of Single CpG Demethylation on the Pattern of DNA-Protein Binding.

Epidermal differentiation is a complex process and its regulation may involve epigenetic factors. Analysis of DNA methylation in 20 selected regions within the epidermal differentiation complex (EDC) gene cluster by targeted next-generation sequencing (NGS) detected no or only minor changes in methylation, mostly slight demethylation, occurring during the course of keratinocyte differentiation. However, a single CpG pair within the exon of the PGLYRP3 gene underwent a pronounced demethylation concomitant with an increase in PGLYRP3 expression. We have employed a DNA-affinity precipitation assay (DAPA) and mass spectrometry to examine changes in the composition of proteins that bind to DNA containing either methylated or unmethylated CpG. We found that the unmethylated probe attracted mostly RNA binding proteins, including splicing factors, which suggests that demethylation of this particular CpG may facilitate PGLYRP3 transcription and/or pre-mRNA splicing.

S100A6 activates EGFR and its downstream signaling in HaCaT keratinocytes.

Epidermal growth factor receptor (EGFR) is a central transmitter of mitogenic signals in epithelial cells; enhanced EGFR activity is observed in many tumors of epithelial origin. S100A6 is a small calcium-binding protein, characteristic mainly of epithelial cells and fibroblasts, strongly implicated in cell proliferation and upregulated in tumors. In this study, using biochemical assays along with immunohistochemical and immunocytochemical analysis of organotypic and standard cultures of HaCaT keratinocytes with S100A6 overexpression or knock-down, we have examined the effect of S100A6 on EGFR activity and downstream signaling. We found that HaCaT cells overexpressing S100A6 had enhanced EGFR, phospho EGFR, and phospho extracellular signal-regulated kinase 1/2 (pERK1/2) staining intensity and level coupled to higher signal transducer and activator of transcription 3 (STAT3) activity. Conversely, S100A6 knockdown cells had impaired EGFR signaling that could be enhanced by addition of recombinant S100A6 to the culture media. Altogether the results show that S100A6 may exert its proproliferative effects through activating EGFR.

Transcriptional regulation of CacyBP/SIP gene and the influence of increased CacyBP/SIP level on gene expression pattern in colorectal cancer HCT116 cells.

The CacyBP/SIP protein is expressed at a particularly high level in brain, spleen, and various tumors. In this work, we have studied transcriptional regulation of the CacyBP/SIP gene and the influence of increased CacyBP/SIP level on gene expression in colorectal cancer HCT116 cells. We have shown that E2F1, EGR1, and CREB transcription factors bind to the CacyBP/SIP gene promoter and stimulate transcription of CacyBP/SIP gene. The role of CREB was further confirmed by the observation that forskolin, a strong activator of CREB phosphorylation/activity, increased CacyBP/SIP gene promoter activity. Moreover, we have shown that CREB dominant negative mutants, CREB133 and KCREB, inhibits CacyBP/SIP promoter activity. To check the biological significance of increased CacyBP/SIP expression/level we have applied RNA microarray analysis and have found that upregulation of CacyBP/SIP entails changes in mRNA level of many genes involved, among others, in immune processes. © 2017 IUBMB Life, 70(1):50-59, 2018.

Current view on cellular function of S100A6 and its ligands, CacyBP/SIP and Sgt1.

S100A6, a calcium binding protein, whose gene was first identified as growth inducible one, has been linked to the process of cell proliferation and growth related phenomena ever since. While the structure and Ca2+ binding kinetics of S100A6 are rather well established the mechanism of its action has only recently begun to be elucidated. It is nonetheless evident that S100A6 exerts its biological role by interacting with a wide range of proteins ligands, many of which have been identified in our laboratory. Our research concentrates on two S100A6 ligands, CacyBP/SIP and Sgt1, which in turn possess their own interactomes. The imposing list of S100A6-interacting proteins indicates that together with its ligands it is a component of an extended network of cellular interactions and may be involved not only in cell proliferation but also in many other processes, of which cell differentiation and response to stress seem to be best documented.

[Rare diseases with epigenetic background]. / Choroby rzadkie o podlozu epigenetycznym.

Rare diseases with epigenetic background arise due to dysregulation of factors/processes that control epigenetic modifications of chromatin and miRNA level. They are usually caused by point mutations or chromosomal aberrations, such as deletions, which occur de novo during early embryonic development. They represent a heterogeneous group of multisystem diseases that mostly affect the nervous system and account for intellectual disability, mild to severe, of affected people. Studies on animal models not only provide a better insight into the molecular mechanisms of the observed anomalies and allow us to causally link the initial alteration in the genome with disease symptoms, but also deliver invaluable data that facilitate the design of effective therapies. Patients suffering from these diseases should receive comprehensive medical care, undergo adequate behavioral and/or occupational therapies, and have access to advanced treatment methods. This work provides information on typical symptoms, molecular basis and the current state of knowledge about selected rare diseases with epigenetic background.

P53 Dysfunction in Neurodegenerative Diseases - The Cause or Effect of Pathological Changes?

Neurodegenerative diseases are a heterogeneous, mostly age-associated group of disorders characterized by progressive neuronal loss, the most prevalent being Alzheimer disease. It is anticipated that, with continuously increasing life expectancy, these diseases will pose a serious social and health problem in the near feature. Meanwhile, however, their etiology remains largely obscure even though all possible novel clues are being thoroughly examined. In this regard, a concept has been proposed that p53, as a transcription factor controlling many vital cellular pathways including apoptosis, may contribute to neuronal death common to all neurodegenerative disorders. In this work, we review the research devoted to the possible role of p53 in the pathogenesis of these diseases. We not only describe aberrant changes in p53 level/activity observed in CNS regions affected by particular diseases but, most importantly, put special attention to the complicated reciprocal regulatory ties existing between p53 and proteins commonly regarded as pathological hallmarks of these diseases, with the ultimate goal to identify the primary element of their pathogenesis.

Regulation of CacyBP/SIP expression by NFAT1 transcription factor.

In this work we have shown that NFAT1 transcription factor is involved in the regulation of CacyBP/SIP expression. We have demonstrated, by applying Western blot, RT-PCR and luciferase assay that the level of CacyBP/SIP increases upon NFAT1 overexpression. Moreover, inhibition or stimulation of NFAT transcriptional activity exerts a corresponding effect on the expression of CacyBP/SIP gene. Furthermore, EMSA and chromatin immunoprecipitation (ChIP) assay have shown that NFAT1 binds to its specific binding sites within the CacyBP/SIP gene. In conclusion, our data have shown for the first time the regulation of CacyBP/SIP gene expression by NFAT1. Since NFAT transcription factors are involved in processes related to immune response, these results indicate potential involvement of CacyBP/SIP in the immune system.

S100A6 - focus on recent developments.

The Ca2+-binding protein, S100A6, belongs to the S100 family. Binding of Ca2+ induces a conformational change, which causes an increase in the overall S100A6 hydrophobicity and allows it to interact with many targets. S100A6 is expressed in different normal tissues and in many tumors. Up to now it has been shown that S100A6 is involved in cell proliferation, cytoskeletal dynamics and tumorigenesis, and that it might have some extracellular functions. In this review, we summarize novel discoveries concerning S100A6 targets, its involvement in cellular signaling pathways, and presence in stem/progenitor cells, extracellular matrix and body fluids of diseased patients.