Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog.

BACKGROUND: A novel fluorescent cAMP analog (8-[Pharos-575]- adenosine-3', 5'-cyclic monophosphate) was characterized with respect to its spectral properties, its ability to bind to and activate three main isoenzymes of the cAMP-dependent protein kinase (PKA-Ialpha, PKA-IIalpha, PKA-IIbeta) in vitro, its stability towards phosphodiesterase and its ability to permeate into cultured eukaryotic cells using resonance energy transfer based indicators, and conventional fluorescence imaging. RESULTS: The Pharos fluorophore is characterized by a Stokes shift of 42 nm with an absorption maximum at 575 nm and the emission peaking at 617 nm. The quantum yield is 30%. Incubation of the compound to RIIalpha and RIIbeta subunits increases the amplitude of excitation and absorption maxima significantly; no major change was observed with RIalpha. In vitro binding of the compound to RIalpha subunit and activation of the PKA-Ialpha holoenzyme was essentially equivalent to cAMP; RII subunits bound the fluorescent analog up to ten times less efficiently, resulting in about two times reduced apparent activation constants of the holoenzymes compared to cAMP. The cellular uptake of the fluorescent analog was investigated by cAMP indicators. It was estimated that about 7 muM of the fluorescent cAMP analog is available to the indicator after one hour of incubation and that about 600 muM of the compound had to be added to intact cells to half-maximally dissociate a PKA type IIalpha sensor. CONCLUSION: The novel analog combines good membrane permeability- comparable to 8-Br-cAMP - with superior spectral properties of a modern, red-shifted fluorophore. GFP-tagged regulatory subunits of PKA and the analog co-localized. Furthermore, it is a potent, PDE-resistant activator of PKA-I and -II, suitable for in vitro applications and spatial distribution evaluations in living cells.

Cryptogenic stroke and small fiber neuropathy of unknown etiology in patients with alpha-galactosidase A -10T genotype.

BACKGROUND: Fabry disease (FD) is a multisystemic disorder with typical neurological manifestations such as stroke and small fiber neuropathy (SFN), caused by mutations of the alpha-galactosidase A (GLA) gene. We analyzed 15 patients carrying the GLA haplotype -10C>T [rs2071225], IVS2-81_-77delCAGCC [rs5903184], IVS4-16A>G [rs2071397], and IVS6-22C>T [rs2071228] for potential neurological manifestations. METHODS AND RESULTS: Patients were retrospectively analyzed for stroke, transient ischemic attack (TIA), white matter lesions (WML) and SFN with neuropathic pain. Functional impact of the haplotype was determined by molecular genetic methods including real-time PCR, exon trapping, promoter deletion constructs and electrophoretic mobility shift assays. Symptomatic -10T allele carriers suffered from stroke, TIA, WML, and SFN with neuropathic pain. Patients' mean GLA mRNA expression level was reduced to ~70% (p < 0.0001) and a dose-dependent effect of the -10T allele on GLA mRNA expression was observed in hemi/homozygous compared to heterozygous patients (p < 0.0001). Molecular analyzes revealed that the -10T allele resulted in a reduced promoter activity and an altered transcription factor binding, while a functional relevance of the co-segregated intronic variants was excluded by exon trapping. CONCLUSIONS: Based on this complementary approach of clinical observation and functional testing, we conclude that the GLA -10T allele could be causal for the observed neurological manifestations. Future studies are needed to clarify whether affected patients benefit from GLA enzyme replacement therapy for end-organ damage prevention.

Tissue-specific differences in the regulation of KIBRA gene expression involve transcription factor TCF7L2 and a complex alternative promoter system.

UNLABELLED: KIBRA has been described as a key regulator of the Hippo signaling pathway, regulating organ size control, cell contact inhibition, cell growth, as well as tumorigenesis and cystogenesis. Since there is scarce information on KIBRA gene expression regulation, we analyzed the molecular basis of tissue-specific KIBRA expression in human kidney epithelial (IHKE, HPCT) and neuroblastoma (SH-SY5Y, SK-SN-SH) cells. We detected four novel and differentially used transcription start sites, two of which positioned in the first intron, generating two novel alternative exons. We identified one constitutively active core promoter (P1a) and three alternative promoters (P1b, P2, and P3), which were exclusively active in kidney cells. Transcription factor 7-like 2 (TCF7L2) selectively activated KIBRA at P1a, P2, and P3 in kidney cells. The two genetic variants -580C>T (p < 0.05) and -1691C>T (p < 0.01) significantly affected the transcriptional activity of the KIBRA core promoter. We propose a novel functional structure of the KIBRA gene and provide detailed insight into molecular cell type-specific KIBRA transcriptional regulation by TCF7L2, the Yes-associated protein 1 and TEA domain family member. Our findings provide a potential basis for future studies on malfunctioning KIBRA regulation in pathophysiological conditions such as cancer development. KEY MESSAGE: KIBRA expression is regulated by three independent, cell type-specific promoters Two novel TSS were located within intron one resulting in two alternative exons TSS utilization is cell type-specific TCF7L2, YAP1, and TEAD are involved in the differential KIBRA expression regulation.

Soluble adenylyl cyclase in vascular endothelium: gene expression control of epithelial sodium channel-α, Na+/K+-ATPase-α/ß, and mineralocorticoid receptor.

The Ca(2+)- and bicarbonate-activated soluble adenylyl cyclase (sAC) has been identified recently as an important mediator of aldosterone signaling in the kidney. Nuclear sAC has been reported to stimulate cAMP response element-binding protein 1 phosphorylation via protein kinase A, suggesting an alternative cAMP pathway in the nucleus. In this study, we analyzed the sAC as a potential modulator of endothelial stiffness in the vascular endothelium. We determined the contribution of sAC to cAMP response element-mediated transcriptional activation in vascular endothelial cells and kidney collecting duct cells. Inhibition of sAC by the specific inhibitor KH7 significantly reduced cAMP response element-mediated promoter activity and affected cAMP response element-binding protein 1 phosphorylation. Furthermore, KH7 and anti-sAC small interfering RNA significantly decreased mRNA and protein levels of epithelial sodium channel-α and Na(+)/K(+)-ATPase-α. Using atomic force microscopy, a nano-technique that measures stiffness and deformability of living cells, we detected significant endothelial cell softening after sAC inhibition. Our results suggest that the sAC is a regulator of gene expression involved in aldosterone signaling and an important regulator of endothelial stiffness. Additional studies are warranted to investigate the protective action of sAC inhibitors in humans for potential clinical use.

Increased monocyte adhesion by endothelial expression of VCAM-1 missense variation in vitro.

OBJECTIVE: In whole genome and single gene analyses, genetic variation at the vascular cell adhesion molecule-1 (VCAM-1) locus has been associated with inflammatory disease and stroke in sickle cell anaemia. In the current work, we investigated the functional impact of VCAM-1 missense variants and their effect on cell-cell adhesion. METHODS AND RESULTS: To determine the functional in vitro relevance of five missense VCAM-1 variants (S318F; T384A; G413A; L555V; I716L), we generated wild type and single variant VCAM-1 forms [318F, 384A, 413A, 555V, 716L] in EA.hy926 endothelial cells. Real-time PCR, western blot and ELISA analyses revealed significant differences in mRNA and protein levels for VCAM-1 variants. Monocytic cell lines THP-1 and U937 showed significantly increased adhesion to endothelial cells overexpressing VCAM-1 forms 318F, 555V and 716L compared to those overexpressing wild type VCAM-1 (p < 0.05). CONCLUSIONS: VCAM-1-dependent cell adhesion to endothelial cells in vitro is significantly increased when expressing VCAM-1 missense mutations 318F, 555V and 716L. The underlying mechanism involves altered VCAM-1 protein levels and function. This observation may be of particular relevance for chronic inflammatory pathophysiologic conditions involving cell-cell adhesion such as atherosclerosis and other proinflammatory conditions.