Cell identity dynamics and insight into insulin secretagogues when employing stem cell-derived islets for disease modeling.

Stem cell-derived islets (SC-islets) are not only an unlimited source for cell-based therapy of type 1 diabetes but have also emerged as an attractive material for modeling diabetes and conducting screening for treatment options. Prior to SC-islets becoming the established standard for disease modeling and drug development, it is essential to understand their response to various nutrient sources in vitro. This study demonstrates an enhanced efficiency of pancreatic endocrine cell differentiation through the incorporation of WNT signaling inhibition following the definitive endoderm stage. We have identified a tri-hormonal cell population within SC-islets, which undergoes reduction concurrent with the emergence of elevated numbers of glucagon-positive cells during extended in vitro culture. Over a 6-week period of in vitro culture, the SC-islets consistently demonstrated robust insulin secretion in response to glucose stimulation. Moreover, they manifested diverse reactivity patterns when exposed to distinct nutrient sources and exhibited deviant glycolytic metabolic characteristics in comparison to human primary islets. Although the SC-islets demonstrated an aberrant glucose metabolism trafficking, the evaluation of a potential antidiabetic drug, pyruvate kinase agonist known as TEPP46, significantly improved in vitro insulin secretion of SC-islets. Overall, this study provided cell identity dynamics investigation of SC-islets during prolonged culturing in vitro, and insights into insulin secretagogues. Associated advantages and limitations were discussed when employing SC-islets for disease modeling.

Glucose Concentration in Regulating Induced Pluripotent Stem Cells Differentiation Toward Insulin-Producing Cells.

The generation of insulin-producing cells from human-induced pluripotent stem cells holds great potential for diabetes modeling and treatment. However, existing protocols typically involve incubating cells with un-physiologically high concentrations of glucose, which often fail to generate fully functional IPCs. Here, we investigated the influence of high (20 mM) versus low (5.5 mM) glucose concentrations on IPCs differentiation in three hiPSC lines. In two hiPSC lines that were unable to differentiate to IPCs sufficiently, we found that high glucose during differentiation leads to a shortage of NKX6.1+ cells that have co-expression with PDX1 due to insufficient NKX6.1 gene activation, thus further reducing differentiation efficiency. Furthermore, high glucose during differentiation weakened mitochondrial respiration ability. In the third iPSC line, which is IPC differentiation amenable, glucose concentrations did not affect the PDX1/NKX6.1 expression and differentiation efficiency. In addition, glucose-stimulated insulin secretion was only seen in the differentiation under a high glucose condition. These IPCs have higher KATP channel activity and were linked to sufficient ABCC8 gene expression under a high glucose condition. These data suggest high glucose concentration during IPC differentiation is necessary to generate functional IPCs. However, in cell lines that were IPC differentiation unamenable, high glucose could worsen the situation.

Acoustic Wave-Induced Stroboscopic Optical Mechanotyping of Adherent Cells.

In this study, a novel, high content technique using a cylindrical acoustic transducer, stroboscopic fast imaging, and homodyne detection to recover the mechanical properties (dynamic shear modulus) of living adherent cells at low ultrasonic frequencies is presented. By analyzing the micro-oscillations of cells, whole populations are simultaneously mechanotyped with sub-cellular resolution. The technique can be combined with standard fluorescence imaging allowing to further cross-correlate biological and mechanical information. The potential of the technique is demonstrated by mechanotyping co-cultures of different cell types with significantly different mechanical properties.

Characterization of Mechanical and Cellular Effects of Rhythmic Vertical Vibrations on Adherent Cell Cultures.

This paper presents an innovative experimental setup that employs the principles of audio technology to subject adherent cells to rhythmic vertical vibrations. We employ a novel approach that combines three-axis acceleration measurements and particle tracking velocimetry to evaluate the setup's performance. This allows us to estimate crucial parameters such as root mean square acceleration, fluid flow patterns, and shear stress generated within the cell culture wells when subjected to various vibration types. The experimental conditions consisted of four vibrational modes: No Vibration, Continuous Vibration, Regular Pulse, and Variable Pulse. To evaluate the effects on cells, we utilized fluorescence microscopy and a customized feature extraction algorithm to analyze the F-actin filament structures. Our findings indicate a consistent trend across all vibrated cell cultures, revealing a reduction in size and altered orientation (2D angle) of the filaments. Furthermore, we observed cell accumulations in the G1 cell cycle phase in cells treated with Continuous Vibration and Regular Pulse. Our results demonstrate a negative correlation between the magnitude of mechanical stimuli and the size of F-actin filaments, as well as a positive correlation with the accumulations of cells in the G1 phase of the cell cycle. By unraveling these analyses, this study paves the way for future investigations and provides a compelling framework for comprehending the intricate cellular responses to rhythmic mechanical stimulation.

A trio of biological rhythms and their relevance in rhythmic mechanical stimulation of cell cultures.

The primary aim of this article is to provide a biological rhythm model based on previous theoretical and experimental findings to promote more comprehensive studies of rhythmic mechanical stimulation of cell cultures, which relates to tissue engineering and regenerative medicine fields. Through an interdisciplinary approach where different standpoints from biology and musicology are combined, we explore some of the core rhythmic features of biological and cellular rhythmic processes and present them as a trio model that aims to afford a basic but fundamental understanding of the connections between various biological rhythms. It is vital to highlight such links since rhythmic mechanical stimulation and its effect on cell cultures are vastly underexplored even though the cellular response to mechanical stimuli (mechanotransduction) has been studied widely and relevant experimental evidence suggests mechanotransduction processes are rhythmic.

The Adenoviral E1B-55k Protein Present in HEK293 Cells Mediates Abnormal Accumulation of Key WNT Signaling Proteins in Large Cytoplasmic Aggregates.

HEK293 cells are one of the most widely used cell lines in research, and HEK293 cells are frequently used as an in vitro model for studying the WNT signaling pathway. The HEK293 cell line was originally established by transfection of human embryonic kidney cells with sheared adenovirus 5 DNA, and it is known that that HEK293 cells stably express the adenoviral E1A and E1B-55k proteins. Here, we show that HEK293 cells display an unexpected distribution of key components of the WNT/ß-catenin signaling pathway where AXIN1, APC, DVL2 and tankyrase are all co-localized in large spherical cytoplasmic aggregates. The cytoplasmic aggregates are enclosed by a narrow layer of the adenoviral E1B-55k protein. The reduction of E1B-55k protein levels leads to the disappearance of the cytoplasmic aggregates thus corroborating an essential role of the E1B-55k protein in mediating the formation of the aggregates. Furthermore, HEK293 cells with reduced E1B-55k protein levels display reduced levels of transcriptional activation of WNT/ß-catenin signaling upon stimulation by the Wnt3A agonist. The demonstrated influence of the E1B-55k protein on the cellular localization of WNT/ß-catenin signaling components and on transcriptional regulation of WNT/ß-catenin signaling asks for caution in the interpretation of data derived from the HEK293 cell line.

Identification of response signatures for tankyrase inhibitor treatment in tumor cell lines.

Small-molecule tankyrase 1 and tankyrase 2 (TNKS1/2) inhibitors are effective antitumor agents in selected tumor cell lines and mouse models. Here, we characterized the response signatures and the in-depth mechanisms for the antiproliferative effect of tankyrase inhibition (TNKSi). The TNKS1/2-specific inhibitor G007-LK was used to screen 537 human tumor cell lines and a panel of particularly TNKSi-sensitive tumor cell lines was identified. Transcriptome, proteome, and bioinformatic analyses revealed the overall TNKSi-induced response signatures in the selected panel. TNKSi-mediated inhibition of wingless-type mammary tumor virus integration site/ß-catenin, yes-associated protein 1 (YAP), and phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT signaling was validated and correlated with lost expression of the key oncogene MYC and impaired cell growth. Moreover, we show that TNKSi induces accumulation of TNKS1/2-containing ß-catenin degradasomes functioning as core complexes interacting with YAP and angiomotin proteins during attenuation of YAP signaling. These findings provide a contextual and mechanistic framework for using TNKSi in anticancer treatment that warrants further comprehensive preclinical and clinical evaluations.

Pancreas-on-a-Chip Technology for Transplantation Applications.

PURPOSE OF REVIEW: Human pancreas-on-a-chip (PoC) technology is quickly advancing as a platform for complex in vitro modeling of islet physiology. This review summarizes the current progress and evaluates the possibility of using this technology for clinical islet transplantation. RECENT FINDINGS: PoC microfluidic platforms have mainly shown proof of principle for long-term culturing of islets to study islet function in a standardized format. Advancement in microfluidic design by using imaging-compatible biomaterials and biosensor technology might provide a novel future tool for predicting islet transplantation outcome. Progress in combining islets with other tissue types gives a possibility to study diabetic interventions in a minimal equivalent in vitro environment. Although the field of PoC is still in its infancy, considerable progress in the development of functional systems has brought the technology on the verge of a general applicable tool that may be used to study islet quality and to replace animal testing in the development of diabetes interventions.

Tankyrase inhibition sensitizes melanoma to PD-1 immune checkpoint blockade in syngeneic mouse models.

The development of immune checkpoint inhibitors represents a major breakthrough in cancer therapy. Nevertheless, a substantial number of patients fail to respond to checkpoint pathway blockade. Evidence for WNT/ß-catenin signaling-mediated immune evasion is found in a subset of cancers including melanoma. Currently, there are no therapeutic strategies available for targeting WNT/ß-catenin signaling. Here we show that a specific small-molecule tankyrase inhibitor, G007-LK, decreases WNT/ß-catenin and YAP signaling in the syngeneic murine B16-F10 and Clone M-3 melanoma models and sensitizes the tumors to anti-PD-1 immune checkpoint therapy. Mechanistically, we demonstrate that the synergistic effect of tankyrase and checkpoint inhibitor treatment is dependent on loss of ß-catenin in the tumor cells, anti-PD-1-stimulated infiltration of T cells into the tumor and induction of an IFNγ- and CD8+ T cell-mediated anti-tumor immune response. Our study uncovers a combinatorial therapeutical strategy using tankyrase inhibition to overcome ß-catenin-mediated resistance to immune checkpoint blockade in melanoma.

MEK Inhibition Induces Canonical WNT Signaling through YAP in KRAS Mutated HCT-15 Cells, and a Cancer Preventive FOXO3/FOXM1 Ratio in Combination with TNKS Inhibition.

The majority of colorectal cancers are induced by subsequent mutations in APC and KRAS genes leading to aberrant activation of both canonical WNT and RAS signaling. However, due to induction of feedback rescue mechanisms some cancers do not respond well to targeted inhibitor treatments. In this study we show that the APC and KRAS mutant human colorectal cancer cell line HCT-15 induces canonical WNT signaling through YAP in a MEK dependent mechanism. This inductive loop is disrupted with combined tankyrase (TNKS) and MEK inhibition. RNA sequencing analysis suggests that combined TNKS/MEK inhibition induces metabolic stress responses in HCT-15 cells promoting a positive FOXO3/FOXM1 ratio to reduce antioxidative and cryoprotective systems.

5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin.

BACKGROUND: Development of resistance to 5-fluorouracil (5-FU) is a major problem in treatment of various cancers including pancreatic cancer. In this study, we reveal important resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma. METHODS: 5-FU resistant (5-FUR), epithelial-to-mesenchymal-like sub-clones of the wild type pancreatic cancer cell line Panc03.27 were previously generated in our lab. We investigated the cytotoxic effect of the endosomal/lysosomal-localizing photosensitizer TPCS2a (fimaporfin) combined with light (photochemical treatment, PCT) using MTS viability assay, and used fluorescence microscopy to show localization of TPCS2a and to investigate the effect of photodamage of lysosomes. Flow cytometric analysis was performed to investigate uptake of photosensitizer and to assess intracellular ROS levels. Expression and localization of LAMP1 was assessed using RT-qPCR, western blotting, and structured illumination microscopy. MTS viability assay was used to assess the effect of combinations of 5-FU, chloroquine (CQ), and photochemical treatment. Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS2a and anti-CD105-saporin was assessed using microscopy. Lastly, the MTS assay was used to investigate cytotoxic effects of photochemical internalization (PCI) of the anti-CD105-immunotoxin. RESULTS: The 5-FUR cell lines display hypersensitivity to PCT, which was linked to increased uptake of TPCS2a, altered lysosomal distribution, lysosomal photodamage and increased expression of the lysosomal marker LAMP-1 in the 5-FUR cells. We show that inhibition of autophagy induced by either chloroquine or lysosomal photodamage increases the sensitivity to 5-FU in the resistant cells. The three 5-FUR sub-clones overexpress Endoglin (CD105). Treatment with the immunotoxin anti-CD105-saporin alone significantly reduced the viability of the CD105-expressing 5-FUR cells, whereas little effect was seen in the CD105-negative non-resistant parental cancer cell lines. Strikingly, using the intracellular drug delivery method photochemical internalization (PCI) by combining light-controlled activation of the TPCS2a with nanomolar levels of CD105-saporin resulted in strong cytotoxic effects in the 5-FUR cell population. CONCLUSION: Our findings suggested that autophagy is an important resistance mechanism against the chemotherapeutic drug 5-FU in pancreatic cancer cells, and that inhibition of the autophagy process, either by CQ or lysosomal photodamage, can contribute to increased sensitivity to 5-FU. For the first time, we demonstrate the promise of PCI-based targeting of CD105 in site-specific elimination of 5-FU resistant pancreatic cancer cells in vitro. In conclusion, PCI-based targeting of CD105 may represent a potent anticancer strategy and should be further evaluated in pre-clinical models.

Discovery of a Novel Series of Tankyrase Inhibitors by a Hybridization Approach.

A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor 16 displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC50 values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker.

Expression profiling of wild type and ß-catenin gene disrupted human BxPC-3 pancreatic adenocarcinoma cells.

To study the role of WNT/ß-catenin signaling in pancreatic adenocarcinoma, human BxPC-3 cell lines deficient of the central canonical WNT signaling protein ß-catenin were established by using zinc-finger nuclease mediated targeted genomic disruption of the ß-catenin gene (CTNNB1). Comparison of the global transcription levels in wild type cells with two ß-catenin gene disrupted clones identified 85 transcripts that were the most differentially regulated. Gene ontology (GO) term enrichment analysis of these transcripts identified "cell adhesion" as the most significantly enriched GO term. Here we describe the data from the transcription profiling analysis published in the article "Implications of Targeted Genomic Disruption of ß-Catenin in BxPC-3 Pancreatic Adenocarcinoma Cells" [1]. Data have been deposited to the Gene Expression Omnibus (GEO) database repository with the dataset identifier GSE63072.

Implications of targeted genomic disruption of ß-catenin in BxPC-3 pancreatic adenocarcinoma cells.

Pancreatic adenocarcinoma (PA) is among the most aggressive human tumors with an overall 5-year survival rate of <5% and available treatments are only minimal effective. WNT/ß-catenin signaling has been identified as one of 12 core signaling pathways that are commonly mutated in PA. To obtain more insight into the role of WNT/ß-catenin signaling in PA we established human PA cell lines that are deficient of the central canonical WNT signaling protein ß-catenin by using zinc-finger nuclease (ZFN) mediated targeted genomic disruption in the ß-catenin gene (CTNNB1). Five individual CTNNB1 gene disrupted clones (BxPC3ΔCTNNB1) were established from a BxPC-3 founder cell line. Despite the complete absence of ß-catenin, all clones displayed normal cell cycle distribution profiles, overall normal morphology and no elevated levels of apoptosis although increased doubling times were observed in three of the five BxPC3ΔCTNNB1 clones. This confirms that WNT/ß-catenin signaling is not mandatory for long term cell growth and survival in BxPC-3 cells. Despite a normal morphology of the ß-catenin deficient cell lines, quantitative proteomic analysis combined with pathway analysis showed a significant down regulation of proteins implied in cell adhesion combined with an up-regulation of plakoglobin. Treatment of BxPC3ΔCTNNB1 cell lines with siRNA for plakoglobin induced morphological changes compatible with a deficiency in the formation of functional cell to cell contacts. In addition, a re-localization of E-cadherin from membranous in untreated to accumulation in cytoplasmatic puncta in plakoglobin siRNA treated BxPC3ΔCTNNB1 cells was observed. In conclusion we describe in ß-catenin deficient BxPC-3 cells a rescue function for plakoglobin on cell to cell contacts and maintaining the localization of E-cadherin at the cellular surface, but not on canonical WNT signaling as measured by TFC/LEF mediated transcription.

Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols.

Iso-octyl chain-hydroxylated oxysterols were determined in attomoles per 10,000 cells concentrations in 10,000-80,000 cultured pancreatic adenocarcinoma cells, using a sensitive, highly automated nano-LC-ESI-MS-based method. Identified oxysterols included 24S hydroxycholesterol (24S-OHC), 25 hydroxycholesterol (25-OHC), and 27 hydroxycholesterol (27-OHC), while 20S hydroxycholesterol and 22S hydroxycholesterol were not detected. Lower mass limit of quantification was 23 fg (65 amol) for 25-OHC and 27-OHC (100 times lower than our previous method) and 54 fg (135 amol) for 24S-OHC, after derivatization into Girard T hydrazones and online sample cleanup using simplified and robust automatic filtration and filter back flushing solid phase extraction LC/MS/MS. The instrument configuration was easily installed using a commercial nano-LC/MS system. Recoveries in spiked sample were 96, 97, and 77% for 24S-OHC, 25-OHC, and 27-OHC, with within- and between-day repeatabilities of 1-21% and 2-20% relative SD, respectively. The study demonstrates the potential of nano-LC in lipidomics/sterolomics.

High sensitivity measurements of active oxysterols with automated filtration/filter backflush-solid phase extraction-liquid chromatography-mass spectrometry.

Oxysterols are important in numerous biological processes, including cell signaling. Here we present an automated filtration/filter backflush-solid phase extraction-liquid chromatography-tandem mass spectrometry (AFFL-SPE-LC-MS/MS) method for determining 24-hydroxysterol and the isomers 25-hydroxycholesterol and 22S-hydroxycholesterol that enables simplified sample preparation, high sensitivity (~25 pg/mL cell lysis sample) and low sample variability. Only one sample transfer step was required for the entire process of cell lysis, derivatization and determination of selected oxysterols. During the procedure, autoxidation of cholesterol, a potential/common problem using standard analytical methods, was found to be negligible. The reversed phase AFFL-SPE-LC-MS/MS method utilizing a 1mm inner diameter column was validated, and used to determine levels of the oxysterol analytes in mouse fibroblast cell lines SSh-LII and NIH-3T3, and human cancer cell lines, BxPC3, HCT-15 and HCT-116. In BxPC3 cells, the AFFL-SPE-LC-MS/MS method was used to detect significant differences in 24S-OHC levels between vimentin+ and vimentin- heterogenous sub-populations. The methodology also allowed monitoring of significant alterations in 24S-OHC levels upon delivery of the Hedgehog (Hh) antagonist MS-0022 in HCT-116 colorectal carcinoma cell lines.

Cellular responses to targeted genomic sequence modification using single-stranded oligonucleotides and zinc-finger nucleases.

Single-stranded oligonucleotides (ssODNs) and zinc-finger nucleases (ZFNs) are two approaches that are being pursued to achieve sequence specific genome modification. ZFNs induce high rates of homologous recombination (HR) between the target sequence and a given donor by introducing site-specific genomic double-strand breaks (DSBs). The mode of action that is used by ssODNs remains largely unknown, but may involve genomic integration of the ssODNs. In this work, cellular responses following ssODN and ZFN mediated correction of a genomic reporter gene have been investigated in human cells. Comparison of the cell cycle distribution of corrected cells following ssODN or ZFN exposure, established that ssODN corrected cells were arrested in the late S and G2/M cell cycle phases, while ZFN corrected cells displayed normal cell cycle profiles. We demonstrate that after ssODN mediated gene correction, phosphorylation of the damage sensor protein H2AX could be observed in 5.8% and 29% of the corrected cells, using a single copy and a multi copy reporter, respectively. When using the ZFN strategy in a single copy reporter only 1.5% of the corrected cells were positive for gamma-H2AX staining. By direct detection of genomic DSBs we establish that the observed cell cycle arrest following ssODN mediated gene correction could be associated with the presence of unrepaired genomic DSBs. Lastly, we establish that although a mutant cellular mismatch repair (MMR) system as expected enhanced ssODN mediated gene correction, the capacity of the ssODN corrected cells to proliferate was not influenced by the MMR system. In conclusion gene correction by means of the ssODN strategy leads to activation of DNA damage signalling and cell cycle arrest due to formation of unrepaired genomic DSBs in a high proportion of the corrected cells. On the contrary, cells corrected using ZFNs displayed normal cell cycle distribution and lower rates of DNA damage.

Genomic sequence correction by single-stranded DNA oligonucleotides: role of DNA synthesis and chemical modifications of the oligonucleotide ends.

BACKGROUND: Single-stranded oligonucleotides (ssODN) can induce site-specific genetic alterations in selected mammalian cells, but the involved mechanisms are not known. METHODS: We corroborate the potential of genomic sequence correction by ssODN using chromosomally integrated mutated enhanced green fluorescent protein (mEGFP) reporter genes in CHO cell lines. The role of integration site was studied in a panel of cell clones with randomly integrated reporters and in cell lines with site-specific single copy integration of the mEGFP reporter in opposite orientations. Involvement of end modification was examined on ssODN with unprotected or phosphorothioate (PS) protected ends. Also ssODN containing octyl or hexaethylene glycol (HEG) end blocking groups were tested. The significance of DNA synthesis was investigated by cell cycle analysis and by the DNA polymerases alpha, delta and epsilon inhibitor aphidicolin. RESULTS: Correction rates of up to 5% were observed upon a single transfection of ssODN. Independent of the mEGFP chromosomal integration site and of its orientation towards the replication fork, antisense ssODN were more effective than sense ssODN. When ssODN ends were blocked by either octyl or HEG groups, correction rates were reduced. Finally, we demonstrate a dependence of the process on DNA synthesis. CONCLUSIONS: We show that, on a chromosomal level, the orientation of the replication fork towards the targeted locus is not central in the strand bias of ssODN-based targeted sequence correction. We demonstrate the importance of accessible ssODN ends for sequence alteration. Finally, we provide evidence for the involvement of DNA synthesis in the process.

A role of the TATA box and the general co-activator hTAF(II)130/135 in promoter-specific trans-activation by simian virus 40 small t antigen.

The small t antigen (st-ag) of simian virus 40 can exert pleiotropic effects on biological processes such as DNA replication, cell cycle progression and gene expression. One possible mode of achieving these effects is through stimulation of NFkappaB-responsive genes encoding growth factors, cytokines, transcription factors and cell cycle regulatory proteins. Indeed, a previous study has shown that st-ag enhanced NFkappaB-mediated transcription. This study demonstrates that promoters possessing a consensus TATA box (i.e. TATAAAAG) in the context of either NFkappaB- or Sp1-binding sites are trans-activated by st-ag. Overexpressing the general transcription factor hTAF(II)130/135, but not hTAF(II)28 or hTAF(II)80, stimulated the activity of promoters in a consensus TATA box-dependent mode. Converting the consensus TATA motif into a non-consensus TATA box strongly impaired activation by st-ag and hTAF(II)130/135. Conversely, mutating a non-consensus TATA motif into the consensus TATA box rendered the mutated promoter inducible by st-ag and hTAF(II)130/135. Mutation of the TATA box had no effect on TNFalpha- or RelA/p65-mediated induction of NFkappaB-responsive promoters, indicating a specific st-ag effect on hTAF(II)130/135. St-ag stimulated the intrinsic transcriptional activity of hTAF(II)130/135. Substitutions in the conserved HPDKGG motif in the N-terminal region or a mutation that impaired the interaction with protein phosphatase 2A abrogated the ability of st-ag to activate hTAF(II)130/135-mediated transcription. These results indicate that trans-activation of promoters by st-ag may depend on a consensus TATA motif and suggest that such promoters recruit the general transcription factor hTAF(II)130/135.

Activation of the coactivator four-and-a-half-LIM-only protein FHL2 and the c-fos promoter through inhibition of protein phosphatase 2A.

Previous studies have demonstrated that the serine/threonine protein phosphatase 2A (PP2A) can modulate the transcriptional activity of several sequence-specific DNA-binding proteins. However, less is known about the effect of PP2A on the activities of general transcription factors and transcriptional coregulators. Here we describe that the activity of a general coactivator, the four-and-a-half-LIM-only protein 2 (FHL2), is regulated in a PP2A-dependent manner. Specific inhibition of PP2A by simian virus 40 (SV40) small t-antigen (st-ag) stimulated the intrinsic transcriptional activity of FHL2 more than 10-fold, while a st-ag mutant unable to bind PP2A had no effect. Overexpression of the B56 subunits alpha, beta, and gamma1 of PP2A impaired the induction of FHL2 by st-ag. FHL2 functioned as a coactivator for CREB-mediated transcription, and inactivation of PP2A further increased FHL2-induced CREB-directed transcription. Overexpression of FHL2 readily enhanced the transcription of the luciferase reporter gene driven by the c-fos promoter, and inhibition of PP2A further stimulated FHL2-induced transactivation of this promoter. These results suggest that dephosphorylation of the general coactivator FHL2 may represent a novel mechanism by which PP2A modulates the transcription of FHL2-responsive genes.