Michael Fähling

Phorbol-ester mediated suppression of hASH1 synthesis: multiple ways to keep the level down

Human achaete-scute homolog-1 (hASH1), encoded by the human ASCL1 gene, belongs to the family of basic helix-loop-helix transcription factors. hASH1 and its mammalian homolog Mash1 are expressed in the central and peripheral nervous system during development, and promote early neuronal differentiation. Furthermore, hASH1 is involved in the specification of neuronal subtype identities. Misexpression of the transcription factor is correlated with a variety of tumors, including lung cancer and neuroendocrine tumors. To gain insights into the molecular mechanisms of hASH1 regulation, we screened for conditions causing changes in hASH1 gene expression rate. We found that treatment of human neuroblastoma-derived Kelly cells with phorbol 12-myristate 13-acetate (PMA) resulted in a fast, strong and long-lasting suppression of hASH1 synthesis. Reporter gene assays with constructs, in which the luciferase activity was controlled either by the ASCL1 promoter or by the hASH1 mRNA untranslated regions (UTRs), revealed a mainly UTR-dependent mechanism. The hASH1 promoter activity was decreased only after 48 h of PMA administration. Our data indicate that different mechanisms acting consecutively at the transcriptional and post-transcriptional level are responsible for hASH1 suppression after PMA treatment. We provide evidence that short term inhibition of hASH1 synthesis is attributed to hASH1 mRNA destabilization, which seems to depend mainly on protein kinase C activity. Under prolonged conditions (48 h), hASH1 suppression is mediated by decreased promoter activity and inhibition of mRNA translation.

Post-Transcriptional Control of the Hypoxic Response by RNA-Binding Proteins and MicroRNAs.

Mammalian gene expression patterns change profoundly in response to low oxygen levels. These changes in gene expression programs are strongly influenced by post-transcriptional mechanisms mediated by mRNA-binding factors: RNA-binding proteins (RBPs) and microRNAs (miRNAs). Here, we review the RBPs and miRNAs that modulate mRNA turnover and translation in response to hypoxic challenge. RBPs such as HuR (human antigen R), PTB (polypyrimidine tract-binding protein), heterogeneous nuclear ribonucleoproteins (hnRNPs), tristetraprolin, nucleolin, iron-response element-binding proteins (IRPs), and cytoplasmic polyadenylation-element-binding proteins (CPEBs), selectively bind to numerous hypoxia-regulated transcripts and play a major role in establishing hypoxic gene expression patterns. MiRNAs including miR-210, miR-373, and miR-21 associate with hypoxia-regulated transcripts and further modulate the levels of the encoded proteins to implement the hypoxic gene expression profile. We discuss the potent regulation of hypoxic gene expression by RBPs and miRNAs and their integrated actions in the cellular hypoxic response.

Adenosine Restores Angiotensin II–Induced Contractions by Receptor-Independent Enhancement of Calcium Sensitivity in Renal Arterioles

Adenosine is coupled to energy metabolism and regulates tissue blood flow by modulating vascular resistance. In this study, we investigated isolated, perfused afferent arterioles of mice, which were subjected to desensitization during repeated applications of angiotensin II. Exogenously applied adenosine restores angiotensin II–induced contractions by increasing calcium sensitivity of the arterioles, along with augmented phosphorylation of the regulatory unit of the myosin light chain. Adenosine restores angiotensin II–induced contractions via intracellular action, because inhibition of adenosine receptors do not prevent restoration, but inhibition of NBTI sensitive adenosine transporters does. Restoration was prevented by inhibition of Rho-kinase, protein kinase C, and the p38 mitogen-activated protein kinase, which modulate myosin light chain phosphorylation and thus calcium sensitivity in the smooth muscle. Furthermore, adenosine application increased the intracellular ATP concentration in LuciHEK cells. The results of the study suggest that restoration of the angiotensin II–induced contraction by adenosine is attributable to the increase of the calcium sensitivity by phosphorylation of the myosin light chain. This can be an important component of vascular control during ischemic and hypoxic conditions. Additionally, this mechanism may contribute to the mediation of the tubuloglomerular feedback by adenosine in the juxtaglomerular apparatus of the kidney.

Splice-specific roles of glycine receptor α3 in the hippocampus

Glycine receptor (GlyR) α3 is involved in vision, and processing of acoustic and nociceptive signals, and RNA editing of GLRA3 transcripts was associated with hippocampal pathophysiology of mesial temporal lobe epilepsy (TLE). However, neither the role of GlyR α3 splicing in hippocampal neurons nor the expression of splice variants have yet been elucidated. We report here that the long (L) splice variant of GlyR α3 predominates in the brain of rodents. Cellular analysis using primary hippocampal neurons and hippocampus cryosections revealed preferential association of synaptic α3L clusters with glutamatergic nerve endings in strata granulare and pyramidale. In primary hippocampal neurons GlyR α3L clusters also preferred glutamatergic nerve endings while α3K was mainly in a diffuse state. Co‐expression of GlyR β subunit with α3L or α3K produced heteromeric receptor clusters and favoured their association with GABAergic terminals. However, heteromeric α3L was still more efficient than heteromeric α3K in associating with glutamatergic nerve endings. To give physiological relevance to these results we have finally analysed GlyR α3 splicing in human hippocampus obtained from patients with intractable TLE. As up‐regulation of α3K occurred at the expense of α3L in TLE patients with a severe course of disease and a high degree of hippocampal damage, our results again involve post‐transcriptional processing of GLRA3 transcripts in the pathophysiology of TLE.

Translational Control of Collagen Prolyl 4-Hydroxylase-α(I) Gene Expression under Hypoxia

Hypoxia is a pro-fibrotic stimulus, which is associated with enhanced collagen synthesis, as well as with augmented collagen prolyl 4-hydroxylase (C-P4H) activity. C-P4H activity is controlled mainly by regulated expression of the α C-P4H subunit. In this study we demonstrate that the increased synthesis of C-P4H-α(I) protein in human HT1080 fibroblasts under long term hypoxia (36 h, 1% oxygen) is controlled at the translational level. This is mediated by an interaction of RNA-binding protein nucleolin (∼64 kDa form) at the 5′- and 3′-untranslated regions (UTR) of the mRNA. The 5′/3′-UTR-dependent mechanism elevates the C-P4H-α(I) expression rate 2.3-fold, and participates in a 5.3-fold increased protein level under long term hypoxia. The interaction of nucleolin at the 5′-UTR occurs directly and depends on the existence of an AU-rich element. Statistical evaluation of the ∼64-kDa nucleolin/RNA interaction studies revealed a core binding sequence, corresponding to UAAAUC or AAAUCU. At the 3′-UTR, nucleolin assembles indirectly via protein/protein interaction, with the help of another 3′-UTR-binding protein, presumably annexin A2. The increased protein level of the ∼64-kDa nucleolin under hypoxia can be attributed to an autocatalytic cleavage of a high molecular weight nucleolin form, without alterations in nucleolin mRNA concentration. Thus, the alteration of translational efficiency by nucleolin, which occurs through a hypoxia inducible factor independent pathway, is an important step in C-P4H-α(I) regulation under hypoxia.

Translational Regulation of the Human Achaete-scute Homologue-1 by Fragile X Mental Retardation Protein

Fragile X syndrome is a common inherited cause of mental retardation that results from loss or mutation of the fragile X mental retardation protein (FMRP). In this study, we identified the mRNA of the basic helix-loop-helix transcription factor human achaete-scute homologue-1 (hASH1 or ASCL1), which is required for normal development of the nervous system and has been implicated in the formation of neuroendocrine tumors, as a new FMRP target. Using a double-immunofluorescent staining technique we detected an overlapping pattern of both proteins in the hippocampus, temporal cortex, subventricular zone, and cerebellum of newborn rats. Forced expression of FMRP and gene silencing by small interference RNA transfection revealed a positive correlation between the cellular protein levels of FMRP and hASH1. A luciferase reporter construct containing the 5′-untranslated region of hASH1 mRNA was activated by the full-length FMRP, but not by naturally occurring truncated FMR proteins, in transient co-transfections. The responsible cis-element was mapped by UV-cross-linking experiments and reporter mutagenesis assays to a (U)10 sequence located in the 5′-untranslated region of the hASH1 mRNA. Sucrose density gradient centrifugation revealed that hASH1 transcripts were translocated into a translationally active polysomal fraction upon transient transfection of HEK293 cells with FMRP, thus indicating translational activation of hASH1 mRNA. In conclusion, we identified hASH1 as a novel downstream target of FMRP. Improved translation efficiency of hASH1 mRNA by FMRP may represent an important regulatory switch in neuronal differentiation.

Cellular oxygen sensing, signalling and how to survive translational arrest in hypoxia

Hypoxia is a consequence of inadequate oxygen availability. At the cellular level, lowered oxygen concentration activates signal cascades including numerous receptors, ion channels, second messengers, as well as several protein kinases and phosphatases. This, in turn, activates trans‐factors like transcription factors, RNA‐binding proteins and miRNAs, mediating an alteration in gene expression control. Each cell type has its unique constellation of oxygen sensors, couplers and effectors that determine the activation and predominance of several independent hypoxia‐sensitive pathways. Hence, altered gene expression patterns in hypoxia result from a complex regulatory network with multiple divergences and convergences. Although hundreds of genes are activated by transcriptional control in hypoxia, metabolic rate depression, as a consequence of reduced ATP level, causes inhibition of mRNA translation. In a multi‐phase response to hypoxia, global protein synthesis is suppressed, mainly by phosphorylation of eIF2‐alpha by PERK and inhibition of mTOR, causing suppression of 5′‐cap‐dependent mRNA translation. Growing evidence suggests that mRNAs undergo sorting at stress granules, which determines the fate of mRNA as to whether being translated, stored, or degraded. Data indicate that translation is suppressed only at ‘free’ polysomes, but is active at subsets of membrane‐bound ribosomes. The recruitment of specific mRNAs into subcellular compartments seems to be crucial for local mRNA translation in prolonged hypoxia. Furthermore, ribosomes themselves may play a significant role in targeting mRNAs for translation. This review summarizes the multiple facets of the cellular adaptation to hypoxia observed in mammals.

Heterogeneous Nuclear Ribonucleoprotein-A2/B1 Modulate Collagen Prolyl 4-Hydroxylase, α (I) mRNA Stability

Collagen prolyl 4-hydroxylase (C-P4H) α-subunit is of regulatory importance in the assembling of C-P4H tetramers, which are necessary for the hydroxylation of procollagen chains. Change in collagen expression by hypoxia or iron diminishment is a significant issue in extracellular matrix remodeling. It was proposed that C-P4H-α (I) is regulated at the posttrancriptional level under these conditions. Here we report that the induction of C-P4H-α (I) in human fibrosarcoma cells HT1080 by the iron chelator 2,2-dipyridyl is predominantly caused by an enhancement of mRNA stability. This effect is mediated by an increased synthesis and binding of heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1, which interacts with a (U)16 element located in the 3′-untranslated region of C-P4H-α (I) mRNA. Luciferase reporter gene assays depending on C-P4H-α (I) 3′-untranslated region and co-transfection with hnRNP-A2/B1 provide evidence that the (U)16 element is necessary and sufficient for posttranscriptional control of C-P4H-α (I) synthesis under the analyzed conditions. Further indication for the significance of hnRNP-A2/B1 in C-P4H-α (I) induction was obtained by micro array experiments. In a data set representing 686 independent physiological conditions, we found a significant positive correlation between hnRNP-A2/B1 and C-P4H-α (I) mRNAs.

Mutation in a primate-conserved retrotransposon reveals a noncoding RNA as a mediator of infantile encephalopathy

The human genome is densely populated with transposons and transposon-like repetitive elements. Although the impact of these transposons and elements on human genome evolution is recognized, the significance of subtle variations in their sequence remains mostly unexplored. Here we report homozygosity mapping of an infantile neurodegenerative disease locus in a genetic isolate. Complete DNA sequencing of the 400-kb linkage locus revealed a point mutation in a primate-specific retrotransposon that was transcribed as part of a unique noncoding RNA, which was expressed in the brain. In vitro knockdown of this RNA increased neuronal apoptosis, consistent with the inappropriate dosage of this RNA in vivo and with the phenotype. Moreover, structural analysis of the sequence revealed a small RNA-like hairpin that was consistent with the putative gain of a functional site when mutated. We show here that a mutation in a unique transposable element-containing RNA is associated with lethal encephalopathy, and we suggest that RNAs that harbor evolutionarily recent repetitive elements may play important roles in human brain development.

Tubular von Hippel-Lindau Knockout Protects against Rhabdomyolysis-Induced AKI

Renal hypoxia occurs in AKI of various etiologies, but adaptation to hypoxia, mediated by hypoxia-inducible factor (HIF), is incomplete in these conditions. Preconditional HIF activation protects against renal ischemia-reperfusion injury, yet the mechanisms involved are largely unknown, and HIF-mediated renoprotection has not been examined in other causes of AKI. Here, we show that selective activation of HIF in renal tubules, through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO), protects from rhabdomyolysis-induced AKI. In this model, HIF activation correlated inversely with tubular injury. Specifically, VHL deletion attenuated the increased levels of serum creatinine/urea, caspase-3 protein, and tubular necrosis induced by rhabdomyolysis in wild-type mice. Moreover, HIF activation in nephron segments at risk for injury occurred only in VHL-KO animals. At day 1 after rhabdomyolysis, when tubular injury may be reversible, the HIF-mediated renoprotection in VHL-KO mice was associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal, as demonstrated by quantitative PCR, pathway enrichment analysis, and immunohistochemistry. In conclusion, a HIF-mediated shift toward improved energy supply may protect against acute tubular injury in various forms of AKI.