Transcription factors Krüppel-like factor 4 and paired box 5 regulate the expression of the Grainyhead-like genes.

Genes from the Grainyhead-like (GRHL) family code for transcription factors necessary for the development and maintenance of various epithelia. These genes are also very important in the development of many types of cancer. However, little is known about the regulation of expression of GRHL genes. Previously, there were no systematic analyses of the promoters of GRHL genes or transcription factors that bind to these promoters. Here we report that the Krüppel-like factor 4 (KLF4) and the paired box 5 factor (PAX5) bind to the regulatory regions of the GRHL genes and regulate their expression. Ectopic expression of KLF4 or PAX5 alters the expression of GRHL genes. In KLF4-overexpressing HEK293 cells, the expression of GRHL1 and GRHL3 genes was upregulated by 32% and 60%, respectively, whereas the mRNA level of GRHL2 gene was lowered by 28% when compared to the respective controls. The levels of GRHL1 and GRHL3 expression were decreased by 30% or 33% in PAX5-overexpressing HEK293 cells. The presence of minor frequency allele of single nucleotide polymorphism rs115898376 in the promoter of the GRHL1 gene affected the binding of KLF4 to this site. The evidence presented here suggests an important role of KLF4 and PAX5 in the regulation of expression of GRHL1-3 genes.

Recent Discoveries on the Involvement of Krüppel-Like Factor 4 in the Most Common Cancer Types.

Krüppel-like factor 4 (KLF4) is a transcription factor highly conserved in evolution. It is particularly well known for its role in inducing pluripotent stem cells. In addition, KLF4 plays many roles in cancer. The results of most studies suggest that KLF4 is a tumor suppressor. However, the functioning of KLF4 is regulated at many levels. These include regulation of transcription, alternative splicing, miRNA, post-translational modifications, subcellular localization, protein stability and interactions with other molecules. Simple experiments aimed at assaying transcript levels or protein levels fail to address this complexity and thus may deliver misleading results. Tumor subtypes are also important; for example, in prostate cancer KLF4 is highly expressed in indolent tumors where it impedes tumor progression, while it is absent from aggressive prostate tumors. KLF4 is important in regulating response to many known drugs, and it also plays a role in tumor microenvironment. More and more information is available about upstream regulators, downstream targets and signaling pathways associated with the involvement of KLF4 in cancer. Furthermore, KLF4 performs critical function in the overall regulation of tissue homeostasis, cellular integrity, and progression towards malignancy. Here we summarize and analyze the latest findings concerning this fascinating transcription factor.

Gonadotropin-releasing hormone-Cu complex (Cu-GnRH) transcriptional activity in vivo in the female rat anterior pituitary gland.

Unlike gonadotropin-releasing hormone (GnRH) analogues characterized by amino acid replacement in decapeptide primary structure, Cu-GnRH molecule preserves the native sequence but contains a Cu2+ ion stably bound to the nitrogen atoms including that of the imidazole ring of His2. Cu-GnRH can operate via cAMP/PKA signalling in anterior pituitary cells, suggesting that it may affect selected gonadotropic network gene transcription in vivo. We analysed pituitary mRNA expression of Egr-1, Nr5a1, and Lhb based on their role in luteinizing hormone (LH) synthesis; and Nos1, Adcyap1, and Prkaca due to their dependence on cAMP/PKA activity. In two independent experiments, ovariectomized rats received intracerebroventricular pulsatile (one pulse/h or two pulses/h over 5 h) microinjections of 2 nM Cu-GnRH; 2 nM antide (GnRH antagonist) + 2 nM Cu-GnRH; 100 nM PACAP6-38 (PACAP receptor antagonist) + 2 nM Cu-GnRH. Relative expression of selected mRNAs was determined by qRT-PCR. LH serum concentration was examined according to RIA. All examined genes responded to Cu-GnRH stimulation with increased transcriptional activity in a manner dependent on pulse frequency pattern. Increased expression of Nr5a1, Lhb, Nos1, Adcyap1, and Prkaca mRNA was observed solely in rats receiving the complex with frequency of two pulses/h over 5 h. Egr-1 transcription was up-regulated for both applied Cu-GnRH pulsatile patterns. The stimulatory effect of Cu-GnRH on gene transcription was dependent on both GnRH receptor and PAC-1 activation. In conclusion, obtained results indicate that Cu-GnRH complex is a GnRH analogue able to induce both IP3/PKC and cAMP/PKA-dependent gonadotrope network gene transcription in vivo.

Neglected Functions of TFCP2/TFCP2L1/UBP1 Transcription Factors May Offer Valuable Insights into Their Mechanisms of Action.

In recent years, the TFCP2 (transcription factor cellular promoter 2)/TFCP2L1 (TFCP2-like 1)/UBP1 (upstream binding protein 1) subfamily of transcription factors has been attracting increasing attention in the scientific community. These factors are very important in cancer, Alzheimer's disease, and other human conditions, and they can be attractive targets for drug development. However, the interpretation of experimental results is complicated, as in principle, any of these factors could substitute for the lack of another. Thus, studying their hitherto little known functions should enhance our understanding of mechanisms of their functioning, and analogous mechanisms might govern their functioning in medically relevant contexts. For example, there are numerous parallels between placental development and cancer growth; therefore, investigating the roles of TFCP2, TFCP2L1, and UBP1 in the placenta may help us better understand their functioning in cancer, as is evidenced by the studies of various other proteins and pathways. Our review article aims to call the attention of the scientific community to these neglected functions, and encourage further research in this field. Here, we present a systematic review of current knowledge of the TFCP2/TFCP2L1/UBP1 subfamily in reproduction, embryonic development, renal function, blood-pressure regulation, brain function, and other processes, where their involvement has not been studied much until now.

Threonine 454 phosphorylation in Grainyhead-like 3 is important for its function and regulation by the p38 MAPK pathway.

The mammalian Grainyhead-like 3 (GRHL3) transcription factor is essential for epithelial development and plays a protective role against squamous cell carcinoma of the skin and of the oral cavity. A single nucleotide polymorphism (SNP) in GRHL3, rs141193530 (p.P455A), is associated with non-melanoma skin cancer in human patients. Moreover, it is known that this SNP, as well as another variant, rs41268753 (p.T454M), are associated with nonsyndromic cleft palate and that rs41268753 negatively affects GRHL3 transcriptional activity. These SNPs are located in adjacent codons of the GRHL3 gene, and the occurrence of either SNP abolishes a putative threonine-proline phosphorylation motif at T454 in the encoded protein. The role of phosphorylation in regulating mammalian GRHL function is currently unknown. In this work we show that GRHL3 is phosphorylated at several residues in a human keratinocyte cell line, among them at T454. This site is essential for the full transcriptional activity of GRHL3. The T454 residue is phosphorylated by p38 MAPK in vitro and activation of p38 signaling in cells causes an increase in GRHL3 activity. The regulation of GRHL3 function by this pathway is dependent on T454, as the substitution of T454 with methionine inhibits the activation of GRHL3. Taken together, our results show that T454 is one of the phosphorylated residues in GRHL3 in keratinocytes and this residue is important for the upregulation of GRHL3 transcriptional activity by the p38 pathway.

TFCP2/TFCP2L1/UBP1 transcription factors in cancer.

The TFCP2/Grainyhead family of transcription factors is divided into two distinct subfamilies, one of which includes the Grainyhead-like 1-3 (GRHL1-3) proteins and the other consists of TFCP2 (synonyms: CP2, LSF, LBP-1c), TFCP2L1 (synonyms: CRTR-1, LBP-9) and UBP1 (synonyms: LBP-1a, NF2d9). Transcription factors from the TFCP2/TFCP2L1/UBP1 subfamily are involved in various aspects of cancer development. TFCP2 is a pro-oncogenic factor in hepatocellular carcinoma, pancreatic cancer and breast cancer, may be important in cervical carcinogenesis and in colorectal cancer. TFCP2 can also act as a tumor suppressor, for example, it inhibits melanoma growth. Furthermore, TFCP2 is involved in epithelial-mesenchymal transition and enhances angiogenesis. TFCP2L1 maintains pluripotency and self-renewal of embryonic stem cells and was implicated in a wide variety of cancers, including clear cell renal cell carcinoma, breast cancer and thyroid cancer. Here we present a systematic review of current knowledge of this protein subfamily in the context of cancer. We also discuss potential challenges in investigating this family of transcription factors. These challenges include redundancies between these factors as well as their interactions with each other and their ability to modulate each other's activity.

[Methods of analysis of protein phosphorylation]. / Metody analizy fosforylacji bialek.

Phosphorylation and dephosphorylation play a fundamental role in most signaling pathways, as these processes can directly regulate various aspects of protein function. It is estimated that there are about 100,000 potential phosphorylation sites in proteins encoded by the human genome and about 30-50% of all proteins in the cell can be phosphorylated, which is directly related to the functions they perform. To determine whether a given protein is phosphorylated, any changes in its mobility caused by this modification are examined during PAGE electrophoresis. Concurrently, tandem mass spectrometry (MS/MS) allows to identify specific phosphorylation sites. The next step involves the prediction (using in silico analysis) which kinases can phosphorylate a specific site in the given protein. Then, in order to verify the information obtained from databases, in vitro and/or in vivo experiments are carried out.

Small intestine motility development in newborn mammals.

Since the beginning of the 20th century, researchers have been working to improve the understanding of gastrointestinal motility. The first major discovery was the observation of a migrating myoelectric complex that turned out to be a universal occurrence among vertebrates. Further inquires resulted in a detailed description of its development during different stages of ontogeny. Some time before that, a cornerstone had been laid for a breakthrough that would come years later. That cornerstone came in the form of interstitial cells of Cajal whose true role could not be discerned until the discovery of a CD117 receptor - their main marker. With the ability to precisely mark interstitial cells of Cajal, a wave of subsequent new experiments and observations connected them to the occurrence of slow waves and allowed an understanding of the mechanism responsible for their generation. Some of these findings suggested that Cajal cells might have a role in the development of several motility disorders thus opening an avenue of research that requires the usage of both traditional and advanced diagnostic methods.

Intracellular mechanisms involved in copper-gonadotropin-releasing hormone (Cu-GnRH) complex-induced cAMP/PKA signaling in female rat anterior pituitary cells in vitro.

The copper-gonadotropin-releasing hormone molecule (Cu-GnRH) is a GnRH analog, which preserves its amino acid sequence, but which contains a Cu(2+) ion stably bound to the nitrogen atoms including that of the imidazole ring of Histidine(2). A previous report indicated that Cu-GnRH was able to activate cAMP/PKA signaling in anterior pituitary cells in vitro, but raised the question of which intracellular mechanism(s) mediated the Cu-GnRH-induced cAMP synthesis in gonadotropes. To investigate this mechanism, in the present study, female rat anterior pituitary cells in vitro were pretreated with 0.1 µM antide, a GnRH antagonist; 0.1 µM cetrorelix, a GnRH receptor antagonist; 0.1 µM PACAP6-38, a PAC-1 receptor antagonist; 2 µM GF109203X, a protein kinase C inhibitor; 50 mM PMA, a protein kinase C activator; the protein kinase A inhibitors H89 (30 µM) and KT5720 (60 nM); factors affecting intracellular calcium activity: 2.5 mM EGTA; 2 µM thapsigargin; 5 µM A23187, a Ca(2+) ionophore; or 10 µg/ml cycloheximide, a protein synthesis inhibitor. After one of the above pretreatments, cells were incubated in the presence of 0.1 µM Cu-GnRH for 0.5, 1, and 3 h. Radioimmunoassay analysis of cAMP confirmed the functional link between Cu-GnRH stimulation and cAMP/PKA signal transduction in rat anterior pituitary cells, demonstrating increased intracellular cAMP, which was reduced in the presence of specific PKA inhibitors. The stimulatory effect of Cu-GnRH on cAMP production was partly dependent on GnRH receptor activation. In addition, an indirect and Ca(2+)-dependent mechanism might be involved in intracellular adenylate cyclase stimulation. Neither activation of protein kinase C nor new protein synthesis was involved in the Cu-GnRH-induced increase of cAMP in the rat anterior pituitary primary cultures. Presented data indicate that conformational changes of GnRH molecule resulting from cooper ion coordination affect specific pharmacological properties of Cu-GnRH molecule including specific pattern of intracellular activity induced by complex in anterior pituitary cells in vitro.