IL-9 Abrogates the Metastatic Potential of Breast Cancer by Controlling Extracellular Matrix Remodeling and Cellular Contractility.

IL-9 is produced by Th9 cells and is classically known as a growth-promoting cytokine. Although protumorigenic functions of IL-9 are described in T cell lymphoma, recently, we and others have reported anti-tumor activities of IL-9 in melanoma mediated by mast cells and CD8+ T cells. However, involvement of IL-9 in invasive breast and cervical cancer remains unexplored. In this study, we demonstrate IL-9-dependent inhibition of metastasis of both human breast (MDA-MB-231 and MCF-7) and cervical (HeLa) tumor cells in physiological three-dimensional invasion assays. To dissect underlying mechanisms of IL-9-mediated suppression of invasion, we analyzed IL-9-dependent pathways of cancer cell metastasis, including proteolysis, contractility, and focal adhesion dynamics. IL-9 markedly blocked tumor cell-collagen degradation, highlighting the effects of IL-9 on extracellular matrix remodeling. Moreover, IL-9 significantly reduced phosphorylation of myosin L chain and resultant actomyosin contractility and also increased focal adhesion formation. Finally, IL-9 suppressed IL-17- and IFN-γ-induced metastasis of both human breast (MDA-MB-231) and cervical (HeLa) cancer cells. In conclusion, IL-9 inhibits the metastatic potential of breast and cervical cancer cells by controlling extracellular matrix remodeling and cellular contractility.

Differential Influence of IL-9 and IL-17 on Actin Cytoskeleton Regulates the Migration Potential of Human Keratinocytes.

T cells mediate skin immune surveillance by secreting specific cytokines and regulate numerous functions of keratinocytes, including migration during homeostasis and disease pathogenesis. Keratinocyte migration is mediated mainly by proteolytic cleavage of the extracellular matrix and/or by cytoskeleton reorganization. However, the cross-talk between T cell cytokines and actomyosin machinery of human primary keratinocytes (HPKs), which is required for cytoskeleton reorganization and subsequent migration, remains poorly examined. In this study, we describe that IL-9 profoundly reduced the actin stress fibers, inhibited contractility, and reduced the cortical stiffness of HPKs, which resulted in inhibition of the migration potential of HPKs in an adhesion- and MMP-independent manner. Similarly, IL-9 inhibited the IFN-γ-induced migration of HPKs by inhibiting the actomyosin machinery (actin stress fibers, contractility, and stiffness). IL-17A increased the actin stress fibers, promoted cellular contractility, and increased proteolytic collagen degradation, resulting in increased migration potential of HPKs. However, IL-9 inhibited the IL-17A-mediated HPKs migration. Mechanistically, IL-9 inhibited the IFN-γ- and IL-17A-induced phosphorylation of myosin L chain in HPKs, which is a major regulator of the actomyosin cytoskeleton. Finally, in addition to HPKs, IL-9 inhibited the migration of A-431 cells (epidermoid carcinoma cells) induced either by IFN-γ or IL-17A. In conclusion, our data demonstrate the influence of T cell cytokines in differentially regulating the actomyosin cytoskeleton and migration potential of human keratinocytes, which may have critical roles in skin homeostasis and pathogenesis of inflammatory diseases as well as skin malignancies.

NF-κB and GATA-Binding Factor 6 Repress Transcription of Caveolins in Bladder Smooth Muscle Hypertrophy.

Caveolins (CAVs) are structural proteins of caveolae that function as signaling platforms to regulate smooth muscle contraction. Loss of CAV protein expression is associated with impaired contraction in obstruction-induced bladder smooth muscle (BSM) hypertrophy. In this study, microarray analysis of bladder RNA revealed down-regulation of CAV1, CAV2, and CAV3 gene transcription in BSM from models of obstructive bladder disease in mice and humans. We identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in mice with obstruction-induced BSM hypertrophy, and in men with benign prostatic hyperplasia. DNA affinity chromatography and chromatin immunoprecipitation assays revealed a greater increase in binding of GATA-binding factor 6 (GATA-6) and NF-κB to their cognate binding motifs on CAV1, CAV2, and CAV3 promoters in obstructed BSM relative to that observed in control BSM. Knockout of NF-κB subunits, shRNA-mediated knockdown of GATA-6, or pharmacologic inhibition of GATA-6 and NF-κB in BSM increased CAV1, CAV2, and CAV3 transcription and promoter activity. Conversely, overexpression of GATA-6 decreased CAV2 and CAV3 transcription and promoter activity. Collectively, these data provide new insight into the mechanisms by which CAV gene expression is repressed in hypertrophied BSM in obstructive bladder disease.

Composite pulse combinations for chirp excitation.

Composite pulses are the efficient method for broadband excitation to get control of the limitations of high field NMR, such as resonance offset effects with constraints on rf power that leads to signal intensity distortion. Phase-modulated chirp pulses are used as ordered composite pulse sequences in this paper as CHORUS sequence in a high-field NMR spectrometer (BRUKER 750 MHz) for broadband excitation. The composite pulse sequence applies chirp pulses with the forward and the reverse sweep mechanisms. A single excitation pulse combines adiabatic and non-adiabatic rotation, explained as a three-phase rotation, which leaves the magnetizing vectors to a non-uniform phase dispersion as a function of the offset frequency. One adiabatic refocusing pulse of the double sweep rate after the excitation pulse cannot satisfactorily compensate for the phase dispersion. Hence, composite self-refocussing CHORUS excitation pulse, with forward, reverse, and their combinations are used to remove the non-uniform phase dispersion generated due to offset resonance frequency. Four such combinations of composite pulses are produced with analytical explanation in this paper. MATLAB simulation results and experimental verification on the BRUKER 750 MHz NMR spectrometer of the composite pulses are also presented in this paper.

Multiomics Analysis and Systems Biology Integration Identifies the Roles of IL-9 in Keratinocyte Metabolic Reprogramming.

IL-9‒producing T cells are present in healthy skin as well as in the cutaneous lesions of inflammatory diseases and cancers. However, the roles of IL-9 in human skin during homeostasis and in the pathogenesis of inflammatory disorders remain obscure. In this study, we examined the roles of IL-9 in metabolic reprogramming of human primary keratinocytes (KCs). High-throughput quantitative proteomics revealed that IL-9 signaling in human primary KCs disrupts the electron transport chain by downregulating multiple electron transport chain proteins. Nuclear magnetic resonance-based metabolomics showed that IL-9 also reduced the production of tricarboxylic acid cycle intermediates in human primary KCs. An integration of multiomics data with systems-level analysis using the constraint-based MitoCore model predicted marked IL-9-dependent effects on central carbohydrate metabolism, particularly in relation to the glycolytic switch. Stable isotope metabolomics and biochemical assays confirmed increased glucose consumption and redirection of metabolic flux toward lactate by IL-9. Functionally, IL-9 inhibited ROS production by IFN-γ and promoted human primary KC survival by inhibiting apoptosis. In conclusion, our data reveal IL-9 as a master regulator of KC metabolic reprogramming and survival.

Blockade of ROCK inhibits migration of human primary keratinocytes and malignant epithelial skin cells by regulating actomyosin contractility.

Actomyosin contractility, crucial for several physiological processes including migration, is controlled by the phosphorylation of myosin light chain (MLC). Rho-associated protein kinase (ROCK) and Myosin light chain kinase (MLCK) are predominant kinases that phosphorylate MLC. However, the distinct roles of these kinases in regulating actomyosin contractility and their subsequent impact on the migration of healthy and malignant skin cells is poorly understood. We observed that blockade of ROCK in healthy primary keratinocytes (HPKs) and epidermal carcinoma cell line (A-431 cells) resulted in loss of migration, contractility, focal adhesions, stress fibres, and changes in morphology due to reduction in phosphorylated MLC levels. In contrast, blockade of MLCK reduced migration, contractile dynamics, focal adhesions and phosphorylated MLC levels of HPKs alone and had no effect on A-431 cells due to the negligible MLCK expression. Using genetically modified A-431 cells expressing phosphomimetic mutant of p-MLC, we show that ROCK dependent phosphorylated MLC controls the migration, focal adhesion, stress fibre organization and the morphology of the cells. In conclusion, our data indicate that ROCK is the major kinase of MLC phosphorylation in both HPKs and A-431 cells, and regulates the contractility and migration of healthy as well as malignant skin epithelial cells.

Hydrogel scaffold with substrate elasticity mimicking physiological-niche promotes proliferation of functional keratinocytes.

High numbers of autologous human primary keratinocytes (HPKs) are required for patients with burns, wounds and for gene therapy of skin disorders. Although freshly isolated HPKs exhibit a robust regenerative capacity, traditional methodology fails to provide a sufficient number of cells. Here we demonstrated a well characterized, non-cytotoxic and inert hydrogel as a substrate that mimics skin elasticity, which can accelerate proliferation and generate higher numbers of HPKs compared to existing tissue culture plastic (TCP) dishes. More importantly, this novel method was independent of feeder layer or any exogenous pharmaceutical drug. The HPKs from the hydrogel-substrate were functional as demonstrated by wound-healing assay, and the expression of IFN-γ-responsive genes (CXCL10, HLADR). Importantly, gene delivery efficiency by a lentiviral based delivery system was significantly higher in HPKs cultured on hydrogels compared with TCP. In conclusion, our study provides the first evidence that cell-material mechanical interaction is enough to provide a rapid expansion of functional keratinocytes that might be used as autologous grafts for skin disorders.

Blockade of Rho-associated protein kinase (ROCK) inhibits the contractility and invasion potential of cancer stem like cells.

Recent studies have implicated the roles of cancer stem like cells (CSCs) in cancer metastasis. However, very limited knowledge exists at the molecular and cellular level to target CSCs for prevention of cancer metastasis. In this study, we examined the roles of contractile dynamics of CSCs in cell invasion and delineated the underlying molecular mechanisms of their distinct cell invasion potential. Using de-adhesion assay and atomic force microscopy, we show that CSCs derived from melanoma and breast cancer cell lines exhibit increased contractility compared to non-CSCs across all tumor types. In addition, CSCs possess increased ECM remodeling capacity as quantified by collagen degradation assay. More importantly, pharmacological blockade of Rho-associated protein kinase completely abolished the contractility and collagen degradation capacity of both CSCs and non-CSCs. In conclusion, our study demonstrates the importance of cell contractility in regulating invasiveness of CSCs and suggests that pharmacological targeting of ROCK pathway represents a novel strategy for targeting both CSCs and bulk population for the treatment of cancer metastasis.