ABSTRACT
Subepithelial fibrosis is a component of the remodeling observed in the bronchial wall of patients diagnosed with asthma. In this process, human bronchial fibroblasts (HBFs) drive the fibroblast-to-myofibroblast transition (FMT) in response to transforming growth factor-ß1 (TGF-ß1), which activates the canonical Smad-dependent signaling. However, the pleiotropic properties of TGF-ß1 also promote the activation of non-canonical signaling pathways which can affect the FMT. In this study we investigated the effect of p38 mitogen-activated protein kinase (MAPK) inhibition by SB203580 on the FMT potential of HBFs derived from asthmatic patients using immunocytofluorescence, real-time PCR and Western blotting methods. Our results demonstrate for the first time the strong effect of p38 MAPK inhibition on the TGF-ß1-induced FMT potential throughout the strong attenuation of myofibroblast-related markers: α-smooth muscle actin (α-SMA), collagen I, fibronectin and connexin 43 in HBFs. We suggest the pleiotropic mechanism of SB203580 on FMT impairment in HBF populations by the diminishing of TGF-ß/Smad signaling activation and disturbances in the actin cytoskeleton architecture along with the maturation of focal adhesion sites. These observations justify future research on the role of p38 kinase in FMT efficiency and bronchial wall remodeling in asthma.
Subject(s)
Asthma/drug therapy , Bronchi/drug effects , Cell Differentiation , Enzyme Inhibitors/pharmacology , Fibroblasts/drug effects , Imidazoles/pharmacology , Pyridines/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Adult , Asthma/enzymology , Asthma/pathology , Bronchi/enzymology , Cells, Cultured , Female , Fibroblasts/enzymology , Humans , Male , Middle Aged , Signal TransductionABSTRACT
The perinucleolar compartment (PNC) is a dynamic, irregularly shaped, and electron-dense nuclear structure that is physically associated with the nucleolus (1). It is found predominantly in transformed cells and various cancer tissues, and rarely in normal cells (1). The components of the PNC described to date include several small RNAs transcribed by RNA polymerase (pol) III, and several RNA binding proteins of which some are primarily implicated in pre-messenger RNA (mRNA) processing (2). The current working model suggests that the PNC is a dynamic functional organelle involved in the metabolism and trafficking of a subset of newly synthesized pol III RNAs in transformed cells. The PNC can be localized and visualized in tissue sections by a immunohistochemical technique using the mouse monoclonal antibody SH54 (3), which specifically recognizes the RNA binding protein PTB (polypyrimidine tract binding protein), which is highly concentrated in the PNC and is used as a marker for PNC detection.The prevalence of PNCs has been found to be correlated with disease progression in breast cancer (3) and in tumors from other tissues, including prostate, colon, ovary, and endometrium (our unpublished studies). PNC prevalence increases with the degree of malignancy and reaches nearly 100% in distant metastases. A high PNC prevalence is associated with poor prognosis (our unpublished studies) (3). In this chapter, we describe methods, which are still under development, for PNC detection and PNC prevalence scoring. Due to the intrinsic limitations of immunocytochemistry using peroxidase assays, the signal intensity can vary from experiment to experiment. Studies are underway to optimize an automated protocol to increase its reproducibility and accuracy.
Subject(s)
Cell Nucleolus/metabolism , Cell Nucleus/metabolism , Immunohistochemistry/methods , Cell Line, Tumor , HeLa Cells , Humans , Models, Biological , Neoplasm Metastasis , Neoplasms/metabolism , Prognosis , RNA/metabolism , RNA, Messenger/metabolism , Reproducibility of ResultsABSTRACT
The perinucleolar compartment (PNC) is a multicomponent nuclear structure enriched with RNAs transcribed by RNA pol III and RNA binding proteins. Studies in cultured cells showed an association between PNC and transformed phenotype. To evaluate the relationship between structure and malignancy in vivo, we examined PNC prevalence (the percentage of cells containing at least one PNC) in normal and cancerous paraffin-embedded breast tissues using immunohistochemistry against a PNC-associated protein. Five hundred nuclei in the most active area of each sample were scored for PNC prevalence. The results show that PNC prevalence significantly correlates with the progression of breast cancer (by the criteria of staging). PNC prevalence in primary tumors, lymph nodes, and distant metastases shows a stepwise increase from a median of 23% in primary tumors to approximately 100% in distant metastases. In addition, univariate and multivariate (controlling for tumor size and grade) analyses show that early-stage patients with invasive ductal carcinomas containing a higher PNC prevalence have a significantly poorer prognosis. These findings link PNC prevalence with the progression of breast cancer in vivo and suggest that PNC-containing cells have metastatic advantages. These findings also show the potential of PNC prevalence as a prognostic marker for breast cancer.
