Rapid signaling reactivation after targeted BRAF inhibition predicts the proliferation of individual melanoma cells from an isogenic population

Cancer cells within tumors display a high degree of phenotypic variability. This variability is thought to allow some of the cells to survive and persist after seemingly effective drug treatments. Studies on vemurafenib, a signaling inhibitor that targets an oncogenic BRAF mutation common in melanoma, suggested that cell-to-cell variation in drug resistance, measured by long-term proliferation, originates from epigenetic differences in gene expression that pre-exist treatment. However, it is still unknown whether reactivation of signaling downstream to the inhibited BRAF, thought to be a key step for resistance, is heterogeneous across cells. While previous studies established that signaling reactivation takes place many hours to days after treatment, they monitored reactivation with bulk-population assays unsuitable for detecting cell-to-cell heterogeneity. We hypothesized that signaling reactivation is heterogeneous and is almost instantaneous for a small subpopulation of resistant cells. We tested this hypothesis by monitoring signaling dynamics at a single-cell resolution and observed that despite highly uniform initial inhibition, roughly 15% of cells reactivated signaling within an hour of treatment. Moreover, by tracking cell lineages over multiple days, we established that these cells indeed proliferated more than neighboring cells, thus establishing that rapid signaling reactivation predicts long-term vemurafenib resistance.

Mutation of Arginine 264 on ERα (Estrogen Receptor Alpha) Selectively Abrogates the Rapid Signaling of Estradiol in the Endothelium Without Altering Fertility

Objective: ERα (estrogen receptor alpha) exerts nuclear genomic actions and also rapid membrane-initiated steroid signaling. The mutation of the cysteine 451 into alanine in vivo has recently revealed the key role of this ERα palmitoylation site on some vasculoprotective actions of 17β-estradiol (E2) and fertility. Here, we studied the in vivo role of the arginine 260 of ERα which has also been described to be involved in its E2-induced rapid signaling with PI-3K (phosphoinositide 3-kinase) as well as G protein in cultured cell lines. Approach and Results: We generated a mouse model harboring a point mutation of the murine counterpart of this arginine into alanine (R264A-ERα). In contrast to the C451A-ERα , the R264A-ERα females are fertile with standard hormonal serum levels and normal control of hypothalamus-pituitary ovarian axis. Although R264A-ERα protein abundance was normal, the well-described membrane ERα–dependent actions of estradiol, such as the rapid dilation of mesenteric arteries and the acceleration of endothelial repair of carotid, were abrogated in R264A-ERα mice. In striking contrast, E2-regulated gene expression was highly preserved in the uterus and the aorta, revealing intact nuclear/genomic actions in response to E2. Consistently, 2 recognized nuclear ERα-dependent actions of E2, namely atheroma prevention and flow-mediated arterial remodeling were totally preserved. Conclusions: These data underline the exquisite role of arginine 264 of ERα for endothelial membrane-initiated steroid signaling effects of E2 but not for nuclear/genomic actions. This provides the first model of fertile mouse with no overt endocrine abnormalities with specific loss-of-function of rapid ERα signaling in vascular functions.

Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics

Wounding is caused by abiotic and biotic factors and triggers complex short- and long-term responses at the local and systemic level. These responses are under the control of complex signaling pathways, which are still poorly understood. Here, we show that the rapid activation of MKK4/5-MPK3/6 by wounding is independent of jasmonic acid (JA) signaling and that, contrary to what happens in tobacco, this fast module does not control wound-triggered JA accumulation in Arabidopsis. We also demonstrate that a second MAPK module, constituted by MKK3 and the clade-C MAPKs MPK1/2/7, is activated by wounding in an independent manner. We provide evidence that the activation of this MKK3-MPK1/2/7 module occurs mainly through wound-induced JA production via the transcriptional regulation of upstream clade-III MAP3Ks and particularly MAP3K14. We show that mkk3 mutant plants are more susceptible to the larvae of the generalist lepidopteran herbivore Spodoptera littoralis, indicating that the MKK3-MPK1/2/7 module is involved in counteracting insect feeding.One sentence summaryWounding induces the parallel activation of a rapid signaling module (MKK4/5-MPK3/6) and a JA-dependent slow one (MAP3K14-MKK3-MPK1/2/7/14) to restrict insect feeding.

Early transcriptomic responses associated with the membrane-initiated action of cortisol in the skeletal muscle of rainbow trout (Oncorhynchus mykiss)

Cortisol is a critical neuroendocrine regulator of the stress response in fish. Cortisol practically affects all tissues by interacting with an intracellular receptor and modulating target gene expression. However, cortisol also interacts with components of the plasma membrane in a nongenomic process that activates rapid signaling. Until now, the implication of this novel cortisol signaling for the global transcriptional response has not been explored. In the present work, we evaluated the effects of the membrane-initiated actions of cortisol on the in vivo transcriptome of rainbow trout ( Oncorhynchus mykiss) skeletal muscle. RNA-Seq analyses were performed to examine the transcriptomic changes in rainbow trout stimulated by physiological concentrations of cortisol and cortisol coupled with bovine serum albumin (cortisol-BSA), a membrane-impermeable analog of cortisol. A total of 660 million paired-ends reads were generated. Reads mapped onto the reference genome revealed that 1,737; 897; and 1,012 transcripts were differentially expressed after 1, 3, and 9 h of cortisol-BSA treatment, respectively. Gene Ontology analysis showed that this novel action of cortisol modulates several biological processes, such as mRNA processing, ubiquitin-dependent protein catabolic processes, and transcription regulation. In addition, a KEGG analysis revealed that focal adhesion was the main signaling pathway that was upregulated at all the times tested. Taking these results together, we propose that the membrane-initiated cortisol action contributes significantly in the regulation of stress-mediated gene expression.

SRXN1 Is Necessary for Resolution of GnRH-Induced Oxidative Stress and Induction of Gonadotropin Gene Expression

A defining characteristic of the hypothalamus-pituitary-gonad reproductive endocrine axis is the episodic secretion of the pituitary gonadotropin hormones LH and FSH by the anterior pituitary gonadotropes. Hormone secretion is dictated by pulsatile stimulation, with GnRH released by hypothalamic neurons that bind and activate the G protein–coupled GnRH receptor expressed by gonadotropes. Hormone secretion and synthesis of gonadotropins are influenced by the amplitude and frequency of GnRH stimulation; variation in either affects the proportion of LH and FSH secreted and the differential regulation of hormone subunit gene expression. Therefore, proper decoding of GnRH signals is essential for appropriate gonadotropin synthesis and secretion. The GnRH receptor robustly activates downstream signaling cascades to facilitate exocytosis and stimulate gene expression and protein synthesis. It is necessary to rapidly quench signaling to preserve sensitivity and adaptability to changing pulse patterns. Reactive oxygen species (ROS) generated by receptor-activated oxidases fulfill the role of rapid signaling intermediates that facilitate robust and transient signaling. However, excess ROS can be detrimental and, unchecked, can confuse signal interpretation. We demonstrate that sulfiredoxin (SRXN1), an ATP-dependent reductase, is essential for normal responses to GnRH receptor signaling and plays a central role in resolution of ROS induced by GnRH stimulation. SRXN1 expression is mitogen-activated protein kinase dependent, and knockdown reduces Lhb and Fshb glycoprotein hormone subunit mRNA and promoter activity. Loss of SRXN1 leads to increased basal and GnRH-stimulated ROS levels. We conclude that SRXN1 is essential for normal responses to GnRH stimulation and plays an important role in ROS management.