Critically High Carboxyhemoglobin Level following Extensive Hysteroscopic Myomectomy.

Studies have demonstrated that during hysteroscopic myomectomy with bipolar diathermy, carbon monoxide is produced and enters the patient's circulation. However, little is known regarding the immediate or long-term sequelae of transient rises in carboxyhemoglobin levels during hysteroscopic surgery. This paper aims to suggest recommendations for acute evaluation, management, patient counseling, and future research. We present a case of a 36-year-old woman (Gravida 0, Para 0) with abnormal uterine bleeding-leiomyoma and resultant anemia, undergoing hysteroscopic resection of a large submucous myoma. During surgery, the patient was found to have a critically elevated level of carboxyhemoglobin and accompanying electrocardiogram derangements. She was managed with prolonged intubation, 100% O2, and trending of her carboxyhemoglobin levels before extubation. This demonstrates the importance of being cognizant of the potentially toxic gaseous byproducts of bipolar resection and of including intravasation of these byproducts in one's consideration of patient safety during extensive resections. Bipolar hysteroscopic resection of large leiomyomas may result in critically high carboxyhemoglobin levels, which can impair end-organ oxygen delivery with resultant ischemia; the risks of myocardial ischemia should be discussed with the anesthesia team before attempting an extensive resection. Electrocardiogram changes indicative of ischemia should prompt discontinuation of the case. Finally, carboxyhemoglobin poisoning should be included in the differential diagnosis of patients who demonstrate longer-than-expected anesthesia recovery times after bipolar resection of large submucous leiomyomas, and they should be managed with repeat evaluation of carboxyhemoglobin levels, supplemental oxygen, and cardiac monitoring.

p190 RhoGAP promotes contact inhibition in epithelial cells by repressing YAP activity.

ARHGAP35 encoding p190A RhoGAP is a cancer-associated gene with a mutation spectrum suggestive of a tumor-suppressor function. In this study, we demonstrate that loss of heterozygosity for ARHGAP35 occurs in human tumors. We sought to identify tumor-suppressor capacities for p190A RhoGAP (p190A) and its paralog p190B in epithelial cells. We reveal an essential role for p190A and p190B to promote contact inhibition of cell proliferation (CIP), a function that relies on RhoGAP activity. Unbiased mRNA sequencing analyses establish that p190A and p190B modulate expression of genes associated with the Hippo pathway. Accordingly, we determine that p190A and p190B induce CIP by repressing YAP-TEAD-regulated gene transcription through activation of LATS kinases and inhibition of the Rho-ROCK pathway. Finally, we demonstrate that loss of a single p190 paralog is sufficient to elicit nuclear translocation of YAP and perturb CIP in epithelial cells cultured in Matrigel. Collectively, our data reveal a novel mechanism consistent with a tumor-suppressor function for ARHGAP35.

A ßPIX-PAK2 complex confers protection against Scrib-dependent and cadherin-mediated apoptosis.

BACKGROUND: During epithelial morphogenesis, a complex comprising the ßPIX (PAK-interacting exchange factor ß) and class I PAKs (p21-activated kinases) is recruited to adherens junctions. Scrib, the mammalian ortholog of the Drosophila polarity determinant and tumor suppressor Scribble, binds ßPIX directly. Scrib is also targeted to adherens junctions by E-cadherin, where Scrib strengthens cadherin-mediated cell-cell adhesion. Although a role for the Scrib-ßPIX-PAK signaling complex in promoting membrane protrusion at wound edges has been elucidated, a function for this complex at adherens junctions remains unknown. RESULTS: Here, we establish that Scrib targets ßPIX and PAK2 to adherens junctions where a ßPIX-PAK2 complex counterbalances apoptotic stimuli transduced by Scrib and elicited by cadherin-mediated cell-cell adhesion. Moreover, we show that this signaling pathway regulates cell survival in response to osmotic stress. Finally, we determine that in suspension cultures, the Scrib-ßPIX-PAK2 complex functions to regulate anoikis elicited by cadherin engagement, with Scrib promoting and the ßPIX-PAK2 complex suppressing anoikis, respectively. CONCLUSIONS: Our findings demonstrate that the Scrib-ßPIX-PAK2 signaling complex functions as an essential modulator of cell survival when localized to adherens junctions of polarized epithelia. The activity of this complex at adherens junctions is thereby essential for normal epithelial morphogenesis and tolerance of physiological stress. Furthermore, when localized to adherens junctions, the Scrib-ßPIX-PAK2 signaling complex serves as a key determinant of anoikis sensitivity, a pivotal mechanism in tumor suppression. Thus, this work also reveals the need to expand the definition of anoikis to include a central role for adherens junctions.

RSK is a principal effector of the RAS-ERK pathway for eliciting a coordinate promotile/invasive gene program and phenotype in epithelial cells.

The RAS-stimulated RAF-MEK-ERK pathway confers epithelial cells with critical motile and invasive capacities during development, tissue regeneration, and carcinoma progression, often via promoting the epithelial-mesenchymal transition (EMT). Many mechanisms by which ERK exerts this control remain elusive. We demonstrate that the ERK-activated kinase RSK is necessary to induce mesenchymal motility and invasive capacities in nontransformed epithelial and carcinoma cells. RSK is sufficient to induce certain motile responses. Expression profiling analysis revealed that a primary role of RSK is to induce transcription of a potent promotile/invasive gene program by FRA1-dependent and -independent mechanisms. The program enables RSK to coordinately modulate the extracellular environment, the intracellular motility apparatus, and receptors mediating communication between these compartments to stimulate motility and invasion. These findings uncover a mechanism whereby the RAS-ERK pathway controls epithelial cell motility by identifying RSK as a key effector, from which emanate multiple highly coordinate transcription-dependent mechanisms for stimulation of motility and invasive properties.

The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity.

The Fc receptor FcRn traffics immunoglobulin G (IgG) in both directions across polarized epithelial cells that line mucosal surfaces, contributing to host defense. We show that FcRn traffics IgG from either apical or basolateral membranes into the recycling endosome (RE), after which the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis without affecting recycling. Another regulatory component of the RE, Rab11a, is dispensable for transcytosis, but regulates recycling to the basolateral membrane only. None of these proteins affect FcRn trafficking away from lysosomes. Thus, FcRn transcytotic and recycling sorting steps are distinct. These results are consistent with a single structurally and functionally heterogeneous RE compartment that traffics FcRn to both cell surfaces while discriminating between recycling and transcytosis pathways polarized in their direction of transport.

The PIX-GIT complex: a G protein signaling cassette in control of cell shape.

Arf and Rho GTP-binding proteins coordinately regulate membrane dynamics and cytoskeletal rearrangements. The Cdc42/Rac guanine nucleotide exchange factor PIX and the Arf GTPase-activating protein GIT form a stable complex in cells. The PIX-GIT complex functions to integrate signaling among Arf, Cdc42, and Rac proteins in response to cues emanating from integrins, heterotrimeric G proteins, receptor tyrosine kinases, and cell-cell interactions. A concept that emerges from the literature is that the PIX-GIT complex serves as a cassette to elicit changes in cell shape essential for polarized cell responses in a wide range of biological contexts.

GIT2 represses Crk- and Rac1-regulated cell spreading and Cdc42-mediated focal adhesion turnover.

G protein-coupled receptor kinase interactors (GITs) regulate focal adhesion (FA) turnover, cell spreading, and motility through direct interaction with paxillin and the Rac-exchange factor Pak-interacting exchange factor beta (betaPIX). However, it is not clear whether GITs function to activate or repress motility or if the predominant GIT forms, GIT1 and GIT2, serve distinct or redundant roles. Here we demonstrate an obligatory role for endogenous GIT2 in repression of lamellipodial extension and FA turnover by Rac1- and Cdc42-dependent signaling pathways, respectively. Moreover, we show that the SH2-SH3 adaptor protein Crk is an essential target of GIT2 inhibition. Unexpectedly, we find that betaPIX is dispensable for the effects elicited by knockdown of GIT2. Finally, we show that loss of GIT2 is sufficient to induce migration of the nontransformed epithelial cell line MCF10A. These results suggest that inactivation of GIT2 function is a required step for induction of cell motility and that GIT2 may be a target of oncogenic signaling pathways that regulate cell migration.

E2F-dependent histone acetylation and recruitment of the Tip60 acetyltransferase complex to chromatin in late G1.

E2F proteins can either activate or repress transcription. Following mitogenic stimulation, repressive E2F4-p130-histone deacetylase complexes dissociate from, while activating species (E2F1, -2, and -3) associate with, target promoters. Histones H3 and H4 simultaneously become hyperacetylated, but it remains unclear whether this is a prerequisite or a consequence of E2F binding. Here, we show that activating E2F species are required for hyperacetylation of target chromatin in human cells. Overexpression of a dominant-negative (DN) E2F1 mutant in serum-stimulated T98G cells blocked all E2F binding, H4 acetylation, and, albeit partially, H3 acetylation. Target gene activation and S-phase entry were also blocked by DN E2F1. Conversely, ectopic activation of E2F1 rapidly induced H3 and H4 acetylation, demonstrating a direct role for E2F in these events. E2F1 was previously shown to bind the histone acetyltransferases (HATs) p300/CBP and PCAF/GCN5. In our hands, ectopically expressed E2F1 also bound the unrelated HAT Tip60 and induced recruitment of five subunits of the Tip60 complex (Tip60, TRRAP, p400, Tip48, and Tip49) to target promoters in vivo. Moreover, E2F-dependent recruitment of Tip60 to chromatin occurred in late G(1) following serum stimulation. We speculate that the activities of multiple HAT complexes account for E2F-dependent acetylation, transcription, and S-phase entry.

MYC recruits the TIP60 histone acetyltransferase complex to chromatin.

The transcription factor MYC binds specific DNA sites in cellular chromatin and induces the acetylation of histones H3 and H4. However, the histone acetyltransferases (HATs) that are responsible for these modifications have not yet been identified. MYC associates with TRRAP, a subunit of distinct macromolecular complexes that contain the HATs GCN5/PCAF or TIP60. Although the association of MYC with GCN5 has been shown, its interaction with TIP60 has never been analysed. Here, we show that MYC associates with TIP60 and recruits it to chromatin in vivo with four other components of the TIP60 complex: TRRAP, p400, TIP48 and TIP49. Overexpression of enzymatically inactive TIP60 delays the MYC-induced acetylation of histone H4, and also reduces the level of MYC binding to chromatin. Thus, the TIP60 HAT complex is recruited to MYC-target genes and, probably with other other HATs, contributes to histone acetylation in response to mitogenic signals.

Genomic targets of the human c-Myc protein.

The transcription factor Myc is induced by mitogenic signals and regulates downstream cellular responses. If overexpressed, Myc promotes malignant transformation. Myc modulates expression of diverse genes in experimental systems, but few are proven direct targets. Here, we present a large-scale screen for genomic Myc-binding sites in live human cells. We used bioinformatics to select consensus DNA elements (CACGTG or E-boxes) situated in the 5' regulatory region of genes and measured Myc binding to those sequences in vivo by quantitative chromatin immunoprecipitation. Strikingly, most promoter-associated E-boxes showed selective recovery with Myc, unlike non-E-box promoters or E-boxes in bulk genomic DNA. Promoter E-boxes were distributed in two groups bound by Myc at distinct frequencies. The high-affinity group included an estimated 11% of all cellular loci, was highly conserved among different cells, and was bound independently of Myc expression levels. Overexpressed Myc associated at increased frequency with low-affinity targets and, at extreme levels, also with other sequences, suggesting that some binding was not sequence-specific. The strongest DNA-sequence parameter defining high-affinity targets was the location of E-boxes within CpG islands, correlating with an open, preacetylated state of chromatin. Myc further enhanced histone acetylation, with or without accompanying induction of mRNA expression. Our findings point to a high regulatory and biological diversity among Myc-target genes.