Smooth Muscle Myosin Research Articles

Rapid time-stamped analysis of filament motility.

The in vitro motility assay is a valuable tool to understand motor protein mechanics, but existing algorithms are not optimized for accurate time resolution. We propose an algorithm that combines trace detection with a time-stamped analysis. By tracking filament ends, we minimize data loss from overlapping and crossing filaments. A movement trace formed by each filament end is created by time-stamping when the filament either first (filament tip) or last (filament tail) occupies a pixel. A frame number vs. distance curve is generated from this trace, which is segmented into regions by slope to detect stop-and-go movement. We show, using generated mock motility videos, accurate detection of velocity and motile fraction changes for velocities < 0.05 pixels per frame, without manual trace dropping and regardless of filament crossings. Compared with established algorithms we show greatly improved accuracy in velocity and motile fraction estimation, with greatly reduced user effort. We tested two actual motility experiments: (1) adenosine triphosphate (ATP) added to skeletal myosin in rigor; (2) myosin light chain phosphatase (MLCP) added to phasic smooth muscle myosin. Our algorithm revealed previously undetectable features: (1) rapid increase in motile fraction paralleled by a slow increase in velocity as ATP concentration increases; (2) simultaneous reductions in velocity and motile fraction as MLCP diffuses into the motility chamber at very low velocities. Our algorithm surpasses existing algorithms in the resolution of time dependent changes in motile fraction and velocity at a wide range of filament lengths and velocities, with minimal user input and CPU time.

Classifying AML Patients with Inv(16) into High-Risk and Low-Risk Relapsed Patients Based on Peritransplantation Minimal Residual Disease Determined By CBFβ/MYH11 Gene Expression

A subtype of patients with core binding factor acute myeloid leukemia (AML) is characterized by the presence of the presence of inv(16)(p13q22) and rarely by a translocation t(16;16)(p13;q22). The final genetic product is the fusion of the CBFβ gene at 16q22 to the smooth muscle myosin heavy chain (MYH11) at 16p13, which is termed as CBFβ/MYH11 fusion gene. Although this type of AML is considered a favorable cytogenetic subgroup both in NCCN guideline or ELN classification, in particular with the application of high-dose cytarabine-based consolidation chemotherapy regimens, disease relapse remains one of the most important causes leading to death, occurring in around 35% of patients. Thus, for this cohort of patients, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is needed to as salvage treatment to improve the overall survival.It has been reported that when patients achieved hematological remission, the MRD prior to transplantation (pre-MRD) has been demonstrated to be a useful parameter to predict leukemia relapse after allo-HSCT. Evaluating minimal residual disease (MRD) by real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) can quantify the expression level of fusion transcript at different time points along with the treatment. More recently, CBFβ/MYH11 fusion gene has been confirmed to be a good MRD marker and further screening out the patients in high-risk of relapse either during the treatment or after transplantation. However, due to the limit number of patients, there has been no studies about predictive value of CBFβ/MYH11expression after allo-HSCT.Therefore, in this study, we attempted to investigate the impact of pre-MRD determined by RQ-PCR-based CBFβ/MYH11 gene expression on transplant outcomes of AML patients with inv(16)(p13q22) or t(16;16)(p13;q22). Besides, our previous studies has demonstrated that in compared to HLA-identical transplantation, haplo-identical allo-HSCT had superior graft-versus-leukemia (GVL) effect in AML patients with standard-risk. Thus, here we also wanted to know whether the level of CBFβ/MYH11 pretransplant could further divide this form of AML into high-risk and low-risk relapse patients before allo-HSCT.Ninety AML patients with inv(16) in complete remission (CR) were monitored CBFβ/MYH11 transcript before and after allo-HSCT as MRD. A total of 23 patients received HLA-matched sibling donor transplantation (MSDT) and 67 patients received unmanipulated haploidentical hematopoietic stem cell transplantation (haplo-HSCT) were analyzed in this study. Patients were divided into four groups based on the value of CBFβ/MYH11 prior to transplantation (pre-MRD): with negative (group 1)/positive (group 2) pre-MRD before MSDT; with negative (group 3)/positive (group 4) pre-MRD before haplo-HSCT. The results showed that patients in group 2 had the highest cumulative incidence of relapse (2-year CIR, 40.7%), the lowest leukemia-free survival (2-year LFS, 50.8%) and the overall survival (2-year OS, 62.5%) among all four groups. The other three groups of patients had comparable CIR, TRM and OS (P>0.05). The patients were also classified into the other three groups according to CBFβ/MYH11 value of +1 month after transplantation: group 5: pre- and post-transplant MRD were both negative; group 6: the value of post-transplant MRD was lower than 0.2%; group 7: the value of post-transplant MRD was higher than 0.2%. Group 7 had the highest CIR compared to group 5 and 6 (P=0.217 & P=0.001, respectively) and the lowest LFS (P=0.072 & P=0.002, respectively), though the TRM and OS of these three groups were comparable (P>0.05). These results indicated that AML patients with inv(16) were able to be separated into high-risk and low-risk relapse groups based on peritransplant MRD determined by RQ-PCR-based CBFβ/MYH11. Haplo-HSCT might overcome the negative impact of pre-MRD on patient outcomes compared to MSDT. DisclosuresNo relevant conflicts of interest to declare.

Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers

Background and aimsAtherosclerosis is a widespread and complicated disease involving phenotypic modulation and transdifferentiation of vascular smooth muscle cells (SMCs), the predominant cells in aorta, as well as changes in endothelial cells and infiltrating monocytes. Alterations in DNA methylation are likely to play central roles in these phenotypic changes, just as they do in normal differentiation and cancer. MethodsWe examined genome-wide DNA methylation changes in atherosclerotic aorta using more stringent criteria for differentially methylated regions (DMRs) than in previous studies and compared these DMRs to tissue-specific epigenetic features. ResultsWe found that disease-linked hypermethylated DMRs account for 85% of the total atherosclerosis-associated DMRs and often overlap aorta-associated enhancer chromatin. These hypermethylated DMRs were associated with functionally different sets of genes compared to atherosclerosis-linked hypomethylated DMRs. The extent and nature of the DMRs could not be explained as direct effects of monocyte/macrophage infiltration. Among the known atherosclerosis- and contractile SMC-related genes that exhibited hypermethylated DMRs at aorta enhancer chromatin were ACTA2 (aorta α2 smooth muscle actin), ELN (elastin), MYOCD (myocardin), C9orf3 (miR-23b and miR-27b host gene), and MYH11 (smooth muscle myosin). Our analyses also suggest a role in atherosclerosis for developmental transcription factor genes having little or no previous association with atherosclerosis, such as NR2F2 (COUP-TFII) and TBX18. ConclusionsWe provide evidence for atherosclerosis-linked DNA methylation changes in aorta SMCs that might help minimize or reverse the standard contractile character of many of these cells by down-modulating aorta SMC-related enhancers, thereby facilitating pro-atherosclerotic phenotypic changes in many SMCs.

Fractionation of embryonic cardiac progenitor cells and evaluation of their differentiation potential

Mid-gestation mouse ventricles (E11.5) contain a larger number of Nkx2.5+ cardiac progenitor cells (CPCs). The proliferation rates are consistently higher in CPCs compared to myocyte population of developing ventricles. Recent studies suggested that CPCs are an ideal donor cell type for replacing damaged tissue in diseased hearts. Thus, the ability to isolate and expand CPCs from embryos or stem cell cultures could be useful for cell fate studies and regenerative therapies. Since embryonic CPCs possess fewer mitochondria compared to cardiomyocytes, we reasoned that CPCs can be fractionated using a fluorescent mitochondrial membrane potential dye (TMRM) and these cells may retain cardiomyogenic potential even in the absence of cardiomyocytes (CMs). FACS sorting of TMRM stained embryonic ventricular cells indicated that over 99% of cells in TMRM high fraction stained positive for sarcomeric myosin (MF20) and all of them expressed Nkx2.5. Although majority of cells present in TMRM low fraction expressed Nkx2.5, very few cells (~1%) stained positive for MF20. Further culturing of TMRM low cells over a period of 48 h showed a progressive increase in MF20 positive cells. Additional analyses revealed that MF20 negative cells in TMRM low fraction do not express markers for endothelial cells (vWF, CD31) or smooth muscle cells (SM myosin). Treatment of TMRM low cells with known cardiogenic factors DMSO and dynorphin B significantly increased the percentage of MF20+ cells compared to untreated cultures. Collectively, these studies suggest that embryonic CPCs can be separated as a TMRM low fraction and their differentiation potential can be enhanced by exogenous addition of known cardiomyogenic factors.

RSK2 restricts blood vessel diameter

Vascular Biology Phosphorylation of myosin proteins enables smooth muscle cells to contract and cause vasoconstriction. This phosphorylation event has generally been attributed to myosin light chain kinase. However, Artamonov et al. discovered that the kinase RSK2 was activated by intraluminal pressure or stimuli that promote vasoconstriction in resistance arteries and that it phosphorylated smooth muscle myosin. Mice lacking RSK2 had dilated arteries and lower blood pressure than wild-type littermates. RSK2 is a possible drug target for manipulating blood pressure. Sci. Signal. 11 , eaar3924 (2018).

Conjunctival myxoid stromal tumour: a distinctive clinicopathological and immunohistochemical study

Background/aimsTo describe the clinicopathological and immunohistochemical characteristics of 10 patients representing a new entity of benign conjunctival myxoid stromal tumours.MethodsRetrospective review of clinical findings, histopathological and immunohistochemical studies identified 10...

RSK2 contributes to myogenic vasoconstriction of resistance arteries by activating smooth muscle myosin and the Na + /H + exchanger

Smooth muscle contraction is triggered when Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates the regulatory light chain of myosin (RLC20). However, blood vessels from Mlck-deficient mouse embryos retain the ability to contract, suggesting the existence of additional regulatory mechanisms. We showed that the p90 ribosomal S6 kinase 2 (RSK2) also phosphorylated RLC20 to promote smooth muscle contractility. Active, phosphorylated RSK2 was present in mouse resistance arteries under normal basal tone, and phosphorylation of RSK2 increased with myogenic vasoconstriction or agonist stimulation. Resistance arteries from Rsk2-deficient mice were dilated and showed reduced myogenic tone and RLC20 phosphorylation. RSK2 phosphorylated Ser19 in RLC in vitro. In addition, RSK2 phosphorylated an activating site in the Na+/H+ exchanger (NHE-1), resulting in cytosolic alkalinization and an increase in intracellular Ca2+ that promotes vasoconstriction. NHE-1 activity increased upon myogenic constriction, and the increase in intracellular pH was suppressed in Rsk2-deficient mice. In pressured arteries, RSK2-dependent activation of NHE-1 was associated with increased intracellular Ca2+ transients, which would be expected to increase MLCK activity, thereby contributing to basal tone and myogenic responses. Accordingly, Rsk2-deficient mice had lower blood pressure than normal littermates. Thus, RSK2 mediates a procontractile signaling pathway that contributes to the regulation of basal vascular tone, myogenic vasoconstriction, and blood pressure and may be a potential therapeutic target in smooth muscle contractility disorders.

Blebbistatin modulates prostatic cell growth and contrapctility through myosin II signaling.

To investigate the effect of blebbistatin (BLEB, a selective myosin inhibitor) on regulating contractility and growth of prostate cells and to provide insight into possible mechanisms associated with these actions. BLEB was incubated with cell lines of BPH-1 and WPMY-1, and intraprostatically injected into rats. Cell growth was determined by flow cytometry, and in vitro organ bath studies were performed to explore muscle contractility. Smooth muscle (SM) myosin isoform (SM1/2, SM-A/B, and LC17a/b) expression was determined via competitive reverse transcriptase PCR. SM myosin heavy chain (MHC), non-muscle (NM) MHC isoforms (NMMHC-A and NMMHC-B), and proteins related to cell apoptosis were further analyzed via Western blotting. Masson's trichrome staining was applied to tissue sections. BLEB could dose-dependently trigger apoptosis and retard the growth of BPH-1 and WPMY-1. Consistent with in vitro effect, administration of BLEB to the prostate could decrease rat prostatic epithelial and SM cells via increased apoptosis. Western blotting confirmed the effects of BLEB on inducing apoptosis through a mechanism involving MLC20 dephosphorylation with down-regulation of Bcl-2 and up-regulation of BAX and cleaved caspase 3. Meanwhile, NMMHC-A and NMMHC-B, the downstream proteins of MLC20, were found significantly attenuated in BPH-1 and WPMY-1 cells, as well as rat prostate tissues. Additionally, BLEB decreased SM cell number and SM MHC expression, along with attenuated phenylephrine-induced contraction and altered prostate SMM isoform composition with up-regulation of SM-B and down-regulation of LC17a, favoring a faster contraction. Our novel data demonstrate BLEB regulated myosin expression and functional activity. The mechanism involved MLC20 dephosphorylation and altered SMM isoform composition.

The Functional Role of Zinc Finger E Box-Binding Homeobox 2 (Zeb2) in Promoting Cardiac Fibroblast Activation.

Following cardiac injury, fibroblasts are activated and are termed as myofibroblasts, and these cells are key players in extracellular matrix (ECM) remodeling and fibrosis, itself a primary contributor to heart failure. Nutraceuticals have been shown to blunt cardiac fibrosis in both in-vitro and in-vivo studies. However, nutraceuticals have had conflicting results in clinical trials, and there are no effective therapies currently available to specifically target cardiac fibrosis. We have previously shown that expression of the zinc finger E box-binding homeobox 2 (Zeb2) transcription factor increases as fibroblasts are activated. We now show that Zeb2 plays a critical role in fibroblast activation. Zeb2 overexpression in primary rat cardiac fibroblasts is associated with significantly increased expression of embryonic smooth muscle myosin heavy chain (SMemb), ED-A fibronectin and α-smooth muscle actin (α-SMA). We found that Zeb2 was highly expressed in activated myofibroblast nuclei but not in the nuclei of inactive fibroblasts. Moreover, ectopic Zeb2 expression in myofibroblasts resulted in a significantly less migratory phenotype with elevated contractility, which are characteristics of mature myofibroblasts. Knockdown of Zeb2 with siRNA in primary myofibroblasts did not alter the expression of myofibroblast markers, which may indicate that Zeb2 is functionally redundant with other profibrotic transcription factors. These findings add to our understanding of the contribution of Zeb2 to the mechanisms controlling cardiac fibroblast activation.

SRSF6 is upregulated in asthmatic horses and involved in the MYH11 SMB expression.

Smooth muscle has a central role in bronchospasm‐induced airway obstruction in asthma. Alternative mRNA splicing of the smooth muscle myosin heavy chain (myh11) gene produces four different isoforms, one of which (SMB) is characterized by the inclusion of the exon5b, which doubles the smooth muscle cells contraction velocity. Deciphering the regulation of the expression levels of the SMB isoform would represent a major step for the understanding of the triggers and pathways leading to airway smooth muscle contraction in asthma. Our objective was therefore, to study the splicing regulation mechanisms of the exon5b in airway smooth muscle cells. Bioinformatics analysis was performed to identify the cis‐regulatory elements present in the exon5b using HSF finder 3 tool. The expression of the corresponding serine/arginine rich protein (SR) genes thus identified was evaluated by quantitative RT‐PCR (qPCR). SRSF1, SRSF6, and hnRNPA1 cis‐acting elements were identified by in silico analysis of the exon5b sequence as splicing regulator candidates. QPCR analyses showed that SRSF1 and SRSF6 are upregulated in ASM cells from asthmatic horses in exacerbation (n = 5) compared to controls (n = 5). The inhibition of the identified splicing factors by small interfering RNA allowed identifying the regulation of the SMB isoform by SRSF6. Our results implicate for the first time the upregulation of SRSF6 and SRSF1 in the asthmatic ASM cells and indicate that SRSF6 induces the exon5b inclusion. This study provides an important first step for the understanding of the triggers and pathways leading to ASM hypercontraction and identifies a possible new target for asthma.

Warthin-Like Tumor of the Esophagus

Warthin's tumor is the second most common benign neoplasm that occurs within the parotid gland, comprising approximately 14 % of all parotid tumors. Clinically, features are lacking and patients are typically asymptomatic. The occurrence of extra-parotid involvement, particularly within the esophagus has not been previously reported. 72-year old male smoker with previous history of H-pylori gastritis, arteriovenous malformation of the jejunum, and colonic polyps presented to his gastroenterologist for evaluation of melena for two weeks duration. Review of systems was negative and physical exam was benign. Laboratory examination showed normocytic anemia. Patient underwent an upper endoscopy which revealed a nodule within the distal third of the esophagus. Endoscopic ultrasound confirmed the presence of an 14 mm nodule embedded within the muscularis mucosa of the esophagus. The nodule was successfully excised via endoscopic mucosal resection. Immunohistochemical stains of the specimen was positive for CK7 and negative for both CK20 and smooth muscle myosin heavy chain. Cytopathology reported a submucosal oncocytic papillary cystadenoma with a lymphoid stroma, consistent with a Warthin's tumor. The patient underwent endoscopic surveillance one month following the excision with no evidence of recurrence. Warthin's tumor most commonly occur in male smokers around the sixth or seventh decade of life. The pathogenesis of this tumor is highly controversial, but it is believed to arise from malignant transformation of ductal cells within the parotid gland secondary to a toxic exposure. These tumors rarely undergo malignant transformation; however, some have been associated with lymphoproliferative diseases due to the involvement of a reactive lymphoid stroma. Histocytological features of a well-encapsulated cystic mass with a lymphoid stroma surrounded by an oncocytic epithelium with papillary projections is required to make the diagnosis. Our patient's nodule histologically appeared as Warthin's tumor. Whether the same prognostic features can be applied remains to be determined.1836_A Figure 1. This 1.4 cm esophageal sub-mucosal tumor is composed of cystic neoplastic glands (T), lined by a double layer of epithelial cells which exhibit a combined oncocytic and papillary histology with surrounding lymphocytic (L) stroma. These features strikingly differentiate the tumor glands from the normal esophageal mucus glands (black arrow).1836_B Figure 2. The mucus secreting acini (a) of the normal esophageal mucous glands (black arrow) are lined by inconspicuous flattened outer myoepithelial cells and large mucus filled inner cuboidal epithelial cells. The cystic tumor (T) glands are lined by an outer cuboidal basal cell and a larger rounded cells with voluminous finely granular pink cytoplasm (oncocytes).1836_C Figure 3. Endoscopic view of the esophageal mass.

The dominant protein phosphatase PP1c isoform in smooth muscle cells, PP1cβ, is essential for smooth muscle contraction

Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase activities toward the myosin regulatory light chain (RLC). Numerous biochemical and pharmacological studies have investigated the specificity and regulatory activity of smooth muscle myosin light-chain phosphatase (MLCP) bound to myosin filaments and comprised of the regulatory myosin phosphatase target subunit 1 (MYPT1) and catalytic protein phosphatase 1cβ (PP1cβ) subunits. Recent physiological and biochemical evidence obtained with smooth muscle tissues from a conditional MYPT1 knockout suggests that a soluble, MYPT1-unbound form of PP1cβ may additionally contribute to myosin RLC dephosphorylation and relaxation of smooth muscle. Using a combination of isoelectric focusing and isoform-specific immunoblotting, we found here that more than 90% of the total PP1c in mouse smooth muscles is the β isoform. Moreover, conditional knockout of PP1cα or PP1cγ in adult smooth muscles did not result in an apparent phenotype in mice up to 6 months of age and did not affect smooth muscle contractions ex vivo In contrast, smooth muscle-specific conditional PP1cβ knockout decreased contractile force development in bladder, ileal, and aortic tissues and reduced mouse survival. Bladder smooth muscle tissue from WT mice was selectively permeabilized to remove soluble PP1cβ to measure contributions of total (α-toxin treatment) and myosin-bound (Triton X-100 treatment) phosphatase activities toward phosphorylated RLC in myofilaments. Triton X-100 reduced PP1cβ content by 60% and the rate of RLC dephosphorylation by 2-fold. These results are consistent with the selective dephosphorylation of RLC by both MYPT1-bound and -unbound PP1cβ forms in smooth muscle.

Rare morphological appearance of breast carcinoma

Clinical question This is a case of an elderly woman who presented symptomatically and on examination was found to have a multifocal breast cancer. An ultrasound-guided axillary node biopsy was...

Testosterone regulates myosin II isoforms expression and functional activity in the rat prostate.

Benign prostatic hyperplasia (BPH) is mainly caused by increased prostatic smooth muscle (SM) tone and prostatic volume. At the molecular level, SM myosin II (SMM II) and non-muscle myosin II (NMM II) mediate SM tone and cell proliferation while testosterone (T) plays a permissive role in the development of BPH. The novel objective of this study was to elucidate the effects of T on the proliferation and apoptosis of rat prostatic cells and SM contractility as well as related regulatory signaling pathways. Briefly, 36 male rats were divided into three groups (sham-operated, surgically castrated, and castrated with T supplementation). In vitro organ bath studies, competitive RT-PCR, Western-blotting analysis, Masson's trichrome staining, and immunofluorescence staining were performed. Our data showed that castration dramatically increased prostatic SM contractility and SM MHC immunostaining revealed a relatively increased SM cell numbers in the stroma. T deprivation altered prostate SMM II isoform composition with upregulation of SM-B and SM2 but downregulation of LC17a, favoring a faster more phasic-type contraction. Moreover, protein expressions of MLCK, p-MLCP, RhoB, ROCK1, and ROCK2 increased in castrated rats. Meanwhile NMM II heavy chain isoforms A, B, and C (NMMHC-A, B, and C isoforms) were altered by castration which may be linked to decreased cell proliferation and increased apoptosis. Our novel data demonstrated T regulates SMM II and NMM II and their functional activities in rat prostate and T ablation not only decreases prostate size (static component) but also changes the prostatic SM tone (dynamic component).

Dysregulated uterine natural killer cells and vascular remodeling in women with recurrent pregnancy losses.

Angiogenesis and vascular remodeling in secretory endometrium represent one of the crucial steps in pregnancy establishment, for which uterine NK (uNK) cells have an important role. Impairment of these steps may proceed to implantation and instigate initial pathology of recurrent pregnancy losses (RPL). In this study, we aim to investigate vascular development and density of uNK cells in secretory endometrium of women with RPL. Mid-secretory phase endometrial tissues from women with RPL (n=15) and fertile controls (n=7) were investigated. CD56+ and CD16+ uNK cells, CD31+ vascular endothelial cells and smooth muscle myosin (SMM)+ . Vascular smooth muscle cells (VSMC) expressing SMM were investigated using immunohistochemistry and western blot. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) was used as well. CD56+ uNK number was significantly higher in women with RPL compared to controls (P<0.0001). uNK cell density by immunohistochemistry was positively correlated with CD56 mRNA expression by qRT-PCR (r2 =0.43, P=0.0137). The number of blood vessels represented by the expression of either CD31 or SMM was higher in women with RPL as compared to controls (P<0.05 and P<0.0001, respectively), and correlated with the number of uNK cell (r2 =0.18, P<0.04, and r2 =0.65, P<0.0001, respectively). The wall thickness of spiral arteries was significantly higher in women with RPL as compared with that of controls (P=0.0027). Increased uNK cells in mid-secretory endometrium are associated with increased vascularization and defective vascular transformation of spiral arteries in women with RPL.

Discriminating the earliest stages of mammary carcinoma using myoepithelial and proliferative markers.

Mammographic screening has led to increased detection of breast cancer at a pre-invasive state, hence modelling the earliest stages of breast cancer invasion is important in defining candidate biomarkers to predict risk of relapse. Discrimination of pre-invasive from invasive lesions is critically important for such studies. Myoepithelial cells are the barrier between epithelial cells and the surrounding stroma in the breast ductal system. A number of myoepithelial immunohistochemistry markers have been identified and validated in human tissue for use by pathologists as diagnostic tools to distinguish in situ carcinoma from invasive breast cancer. However, robust myoepithelial markers for mouse mammary tissue have been largely under-utilised. Here, we investigated the utility of the myoepithelial markers smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC), cytokeratin-14 (CK14) and p63 to discriminate mammary intraepithelial neoplasia (MIN) from invasive disease in the C57BL/6J MMTV-PyMT transgenic model of mammary carcinoma. We identified that SMMHC and CK14 are retained in early in situ neoplasia and are appropriate markers for distinguishing MIN from invasive disease in this model. Additionally, the proliferation marker Ki67 is a superior marker for differentiating between normal and hyperplastic ducts, prior to the development of MIN. Based on this, we developed a scoring matrix for discriminating normal, hyperplasia, MIN and invasive lesions in this spontaneous mammary tumorigenesis model. This study demonstrates heterogeneous expression of myoepithelial proteins throughout tumour development, and highlights the need to characterise the most appropriate markers in other models of early breast cancer to allow accurate classification of disease state.

A novel human cell culture model to study visceral smooth muscle phenotypic modulation in health and disease.

Adaptation of the smooth muscle cell (SMC) phenotype is essential for homeostasis and is often involved in pathologies of visceral organs (e.g., uterus, bladder, gastrointestinal tract). In vitro studies of the behavior of visceral SMCs under (patho)-physiological conditions are hampered by a spontaneous, uncontrolled phenotypic modulation of visceral SMCs under regular tissue culture conditions. We aimed to develop a new visceral SMC culture model that allows controlled phenotypic modulation. Human uterine SMCs [ULTR and telomerase-immortalized human myometrial cells (hTERT-HM)] were grown to confluency and kept for up to 6 days on regular tissue culture surfaces or basement membrane (BM) matrix-coated surfaces in the presence of 0-10% serum. mRNA and protein expression and localization of SMC-specific phenotype markers and their transcriptional regulators were investigated by quantitative PCR, Western blotting, and immunofluorescence. Maintaining visceral SMCs confluent for 6 days increased α-smooth muscle actin (1.9-fold) and smooth muscle protein 22-α (3.1-fold), whereas smooth muscle myosin heavy chain was only slightly upregulated (1.3-fold). Culturing on a BM matrix-coated surface further increased these proteins and also markedly promoted mRNA expression of γ-smooth muscle actin (15.0-fold), smoothelin (3.5-fold), h-caldesmon (5.2-fold), serum response factor (7.6-fold), and myocardin (8.1-fold). Whereas additional serum deprivation only minimally affected contractile markers, platelet-derived growth factor-BB and transforming growth factor β1 consistently reduced versus increased their expression. In conclusion, we present a simple and reproducible visceral SMC culture system that allows controlled phenotypic modulation toward both the synthetic and the contractile phenotype. This may greatly facilitate the identification of factors that drive visceral SMC phenotypic changes in health and disease.

Endobronchial Smooth Muscle Tumors: A Series of Five Cases Highlighting Pitfalls in Diagnosis.

BackgroundPrimary endobronchial smooth muscle tumors (SMTs), which are extremely rare, include endobronchial leiomyomas and leiomyosarcomas. Clinically, SMTs present with signs and symptoms of bronchial obstruction, and lack specific radiological findings. Thus, histopathological examination is required for accurate diagnosis as well as for tumor grading. We examined the histomorphological and immunohistochemical features of endobronchial SMTs and highlighted pitfalls in diagnosis, particularly when using small biopsies. MethodsCases of primary endobronchial SMTs diagnosed at our Institute over the last 6 years (2012–2017) were retrieved from the departmental archives. Histopathological features and immunohistochemistry performed for establishing the diagnosis were reviewed. ResultsFive cases of SMTs occurring in endobronchial locations were identified. These included three cases of leiomyoma, and two cases of leiomyosarcoma. The age distribution of patients ranged from 13 to 65 years. Leiomyomas showed more consistent staining with smooth muscle markers (smooth muscle actin, desmin, and smooth muscle myosin heavy chain), while tumors of higher grade showed variable, focal staining, leading to erroneous diagnosis, especially on small biopsies. ConclusionsThe diagnosis of endobronchial SMTs relies on histopathological examination, for both confirmation of smooth muscle lineage and determination of the malignant potential of the lesion. Appropriate immunohistochemical panels including more than one marker of smooth muscle differentiation are extremely valuable for differential diagnosis from morphological mimics, which is necessary for instituting appropriate management.

Abstract 5419: HDAC1 regulates RUNX1 activity in inv(16) acute myeloid leukemia

Abstract RUNX1 and CBFβ form a transcription factor dimer that regulates normal hematopoiesis and leukemogenesis. Inversion of chromosome 16 (inv(16)) is one of the most common mutations in acute myeloid leukemia (AML), fusing CBFβ with the gene encoding smooth muscle myosin heavy chain (MYH11). The fusion protein encoded by CBFB-MYH11 (CM), retains the ability to bind to RUNX1, and together they cause changes in gene expression leading to leukemogenesis. Recently, we found that Histone Deacetylase 1 (HDAC1) is part of the RUNX1:CM complex, that all three proteins co-localize on the promoters of target genes, and that HDAC1 is required for target gene expression. By deacetylating histones, HDAC1 can act as a transcriptional repressor. However, HDAC1 can also deacetylate non-histone proteins, which may explain its unexpected role in gene activation. We hypothesized that HDAC1 acts by deacetylating proteins in the RUNX1:CM complex. To test this, we immunoprecipitated the RUNX1:CM complex and probed for acetyl-lysine. The only visible band was at the expected size for RUNX1, suggesting that RUNX1 is the target of HDAC1 activity. Indeed, cells transfected with RUNX1 and HDAC1 showed significantly less acetylation than cells with RUNX1 alone. To test if HDAC1 deacetylates endogenous RUNX1, we treated leukemia cells from CM-expressing knockin mice with the HDAC1 selective inhibitor entinostat. The level of RUNX1 acetylation was significantly increased in treated cells compared to control, providing further support that HDAC1 deacetylates RUNX1. Acetylation at lysines 24 and 43 of RUNX1 was previously shown to increase its activity. To determine the effect of deacetylation on RUNX1 activity, we used the M-CSFR promoter fused to luciferase. Cells transfected with RUNX1, CBFβ, and HDAC1 showed significantly decreased promoter activity compared to cells with RUNX1 and CBFβ. Importantly, in cells expressing RUNX1 acetylation and deacetylation mimetics, promoter activity was unchanged with the addition of HDAC1, indicating that lysines 24 and 43 are the relevant HDAC1 targets. To test if RUNX1 acetylation status affects the activity of the CM complex, we transfected a CM+ cell line with WT or mutated RUNX1 and examined target gene expression. In CM+ cells over-expressing WT RUNX1, we found similar target gene expression as untransfected CM+ cells. However, expression of either of RUNX1 mutant blocked the CM-induced changes in gene expression. This implies that regulation of RUNX1 acetylation is important for the activity of the RUNX1:CM complex. The observation that both RUNX1 mimetics had the same effect on CM activity may imply that alternate rounds of RUNX1 acetylation and deacetylation is required for continuous RUNX1:CM transcriptional activity. This work provides a possible explanation for HDAC1's role in gene activation in inv(16) AML, and may have implications for other RUNX1 dependent leukemias as well. Citation Format: Lisa Richter, Yiqian Wang, Michelle Becker, Jake Williams, R. Katherine Hyde. HDAC1 regulates RUNX1 activity in inv(16) acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5419.

Rho kinase collaborates with p21-activated kinase to regulate actin polymerization and contraction in airway smooth muscle.

The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F-actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine-threonine kinase Pak, which mediates the activation of Cdc42 and neuronal Wiskott-Aldrich syndrome protein (N-WASp). N-WASP transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and Cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F-actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase-inactive mutant of ROCK, ROCK-K121G, in the tissues or by treating them with the ROCK inhibitor H-1152P. Our results show no role for ROCK in the regulation of non-muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue. We found that ROCK regulates airway smooth muscle contraction by mediating activation of p21-activated kinase (Pak), a serine-threonine kinase, to promote actin polymerization. Pak catalyses paxillin phosphorylation on Ser273 and coupling of the GIT1-βPIX-Pak signalling module to paxillin, which activates the guanine nucleotide exchange factor (GEF) activity of βPIX towards Cdc42. Cdc42 is required for the activation of neuronal Wiskott-Aldrich syndrome protein (N-WASp), which transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. Our results demonstrate a novel molecular function for ROCK in the regulation of Pak and Cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle.