Stress-induced isoforms of MDM2 and MDM4 correlate with high-grade disease and an altered splicing network in pediatric rhabdomyosarcoma.

Pediatric rhabdomyosarcoma (RMS) is a morphologically and genetically heterogeneous malignancy commonly classified into three histologic subtypes, namely, alveolar, embryonal, and anaplastic. An issue that continues to challenge effective RMS patient prognosis is the dearth of molecular markers predictive of disease stage irrespective of tumor subtype. Our study involving a panel of 70 RMS tumors has identified specific alternative splice variants of the oncogenes Murine Double Minute 2 (MDM2) and MDM4 as potential biomarkers for RMS. Our results have demonstrated the strong association of genotoxic-stress inducible splice forms MDM2-ALT1 (91.6% Intergroup Rhabdomyosarcoma Study Group stage 4 tumors) and MDM4-ALT2 (90.9% MDM4-ALT2-positive T2 stage tumors) with high-risk metastatic RMS. Moreover, MDM2-ALT1-positive metastatic tumors belonged to both the alveolar (50%) and embryonal (41.6%) subtypes, making this the first known molecular marker for high-grade metastatic disease across the most common RMS subtypes. Furthermore, our results show that MDM2-ALT1 expression can function by directly contribute to metastatic behavior and promote the invasion of RMS cells through a matrigel-coated membrane. Additionally, expression of both MDM2-ALT1 and MDM4-ALT2 increased anchorage-independent cell-growth in soft agar assays. Intriguingly, we observed a unique coordination in the splicing of MDM2-ALT1 and MDM4-ALT2 in approximately 24% of tumor samples in a manner similar to genotoxic stress response in cell lines. To further explore splicing network alterations with possible relevance to RMS disease, we used an exon microarray approach to examine stress-inducible splicing in an RMS cell line (Rh30) and observed striking parallels between stress-responsive alternative splicing and constitutive splicing in RMS tumors.

Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2.

The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser(2113). Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.

Cyclic AMP-Rap1A signaling activates RhoA to induce α(2c)-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells.

Intracellular signaling by the second messenger cyclic AMP (cAMP) activates the Ras-related small GTPase Rap1 through the guanine exchange factor Epac. This activation leads to effector protein interactions, activation, and biological responses in the vasculature, including vasorelaxation. In vascular smooth muscle cells derived from human dermal arterioles (microVSM), Rap1 selectively regulates expression of G protein-coupled α(2C)-adrenoceptors (α(2C)-ARs) through JNK-c-jun nuclear signaling. The α(2C)-ARs are generally retained in the trans-Golgi compartment and mobilize to the cell surface and elicit vasoconstriction in response to cellular stress. The present study used human microVSM to examine the role of Rap1 in receptor localization. Complementary approaches included murine microVSM derived from tail arteries of C57BL6 mice that express functional α(2C)-ARs and mice deficient in Rap1A (Rap1A-null). In human microVSM, increasing intracellular cAMP by direct activation of adenylyl cyclase by forskolin (10 µM) or selectively activating Epac-Rap signaling by the cAMP analog 8-pCPT-2'-O-Me-cAMP (100 µM) activated RhoA, increased α(2C)-AR expression, and reorganized the actin cytoskeleton, increasing F-actin. The α(2C)-ARs mobilized from the perinuclear region to intracellular filamentous structures and to the plasma membrane. Similar results were obtained in murine wild-type microVSM, coupling Rap1-Rho-actin dynamics to receptor relocalization. This signaling was impaired in Rap1A-null murine microVSM and was rescued by delivery of constitutively active (CA) mutant of Rap1A. When tested in heterologous HEK293 cells, Rap1A-CA or Rho-kinase (ROCK-CA) caused translocation of functional α(2C)-ARs to the cell surface (~4- to 6-fold increase, respectively). Together, these studies support vascular bed-specific physiological role of Rap1 and suggest a role in vasoconstriction in microVSM.

Rap1 GTPases: an emerging role in the cardiovasculature.

The Ras related GTPase Rap has been implicated in multiple cellular functions. A vital role for Rap GTPase in the cardiovasculature is emerging from recent studies. These small monomeric G proteins act as molecular switches, coupling extracellular stimulation to intracellular signaling through second messengers. This member of the Ras superfamily was once described as the transformation suppressor with the ability to ameliorate the Ras transformed phenotype; however, further studies uncovered a unique set of guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and effector proteins for Rap suggesting a more sophisticated role for this small GTPase. At least three different second messengers can activate Rap, namely cyclic AMP (cAMP), calcium and diacylglycerol. More recently, an investigation of Rap in the cardiovasculature has revealed multiple pathways of regulation involving Rap in this system. Two closely related isoforms of Rap1 exist, 1a and 1b. Murine genetic models exist for both and have been described. Although thought at first to be functionally redundant, these isoforms have differing roles in the cardiovasculature. The activation of Rap1a and 1b in various cell types of the cardiovasculature leads to alterations in cell attachment, migration and cell junction formation. This review will focus on the role of these Rap1 GTPases in hematopoietic, endothelial, smooth muscle, and cardiac myocyte function, and conclude with their potential role in human disease.

Transcriptional control of human antigen R by bone morphogenetic protein.

Human antigen R (HuR) is an RNA-binding protein with protective activities against cellular stress. This study considers the mechanisms by which HuR transcriptional regulation occurs in renal proximal tubule cells. Under basal conditions, HuR mRNA is expressed in two forms: one that contains a approximately 20-base 5'-untranslated region (UTR) sequence and one that contains a approximately 150-base, G+C-rich 5'-UTR that is inhibitory to translation. Recovery from cellular stresses such as thapsigargin and ATP depletion induced increased expression of the shorter, more translatable transcript and decreased expression of the longer form. Analysis of HuR upstream regions revealed sequences necessary for regulation of the shorter mRNA. Within the long, G+C-rich 5'-UTR exist multiple copies of the alternate Smad 1/5/8-binding motif GCCGnCGC. Recovery from ATP depletion induced increases in Smad 1/5/8 levels; further, gel shift and chromatin immunoprecipitation analyses demonstrated the ability of these Smads to bind to the relevant motif in the HuR 5'-UTR. Transfection of exogenous Smad 1 increased HuR mRNA expression. Finally, HuR mRNA expression driven by the Smad-binding sites was responsive to BMP-7, a protein with known protective effects against ischemic injury in kidney. These data suggest that transcriptional induction of a readily translatable HuR mRNA may be driven by a mechanism known to protect the kidney from injury and provides a novel pathway through which administration of BMP-7 may attenuate renal damage.

Expression and distribution of HuR during ATP depletion and recovery in proximal tubule cells.

Human antigen R (HuR) is a nucleocytoplasmic shuttling protein that binds to and stabilizes mRNAs containing adenine- and uridine-rich elements. Under normal growth conditions, the bulk of HuR is maintained in the nucleus, but under conditions of cell stress, HuR may become more prevalent in the cytosol, where it can stabilize mRNA and regulate gene expression. We have studied the behavior of HuR in LLC-PK1 proximal tubule cells subjected to ATP depletion and recovery. ATP depletion resulted in detectable net movement of HuR out of the nucleus, followed by net movement of HuR back into the nucleus on reversion to normal growth medium. In addition, HuR protein levels increased during energy depletion. This increase was inhibited by cycloheximide and was independent of HuR mRNA levels, since no change was noted in the quantity of HuR transcript. In contrast, recovery in normal growth medium resulted in increased HuR mRNA, while protein levels decreased to baseline. This suggested a mechanism by which previously injured cells maintained normal levels of HuR but were primed to rapidly translate increased amounts of protein on subsequent insults. Indeed, a second round of ATP depletion resulted in heightened HuR protein translation at a rate more rapid than during the first insult. Additionally, the second insult produced increased HuR levels in the cytoplasm while still maintaining high amounts in the nucleus, indicating that nuclear export may not be required on subsequent insults. These results suggest a role for HuR in protecting kidney epithelia from injury during ischemic stress.