Sheryl L. White

Expression of the Smooth Muscle Myosin Heavy Chain Gene Is Regulated by a Negative-acting GC-rich Element Located between Two Positive-acting Serum Response Factor-binding Elements

To identify cis- and trans-acting factors that regulate smooth muscle-specific gene expression, we studied the smooth muscle myosin heavy chain gene, a rigorous marker of differentiated smooth muscle. A comparison of smooth muscle myosin heavy chain promoter sequences from multiple species revealed the presence of a highly conserved 227-base pair domain (nucleotides −1321 to −1095 in rat). Results of a deletion analysis of a 4.3-kilobase pair segment of the rat promoter (nucleotides −4220 to +88) demonstrated that this domain was necessary for maximal transcriptional activity in smooth muscle cells. Gel-shift analysis and site-directed mutagenesis demonstrated that one true CArG and another CArG-like element contained within this domain were both recognized by the serum response factor and were both required for the positive activity attributable to this domain. Additional studies demonstrated that mutation of a GC-rich sequence within the 227-base pair conserved domain resulted in a nearly 100% increase in transcriptional activity. Gel-shift analysis showed that this GC-rich repressor element was recognized by both Sp1 and Sp3. These data demonstrate that transcriptional control of the smooth muscle myosin heavy chain gene is highly complex, involving both negative and positive regulatory elements, including CArG sequences found in the promoters of multiple smooth muscle differentiation marker genes.

Therapies for bleomycin induced lung fibrosis through regulation of TGF‐β1 induced collagen gene expression

This review describes normal and abnormal wound healing, the latter characterized by excessive fibrosis and scarring, which for lung can result in morbidity and sometimes mortality. The cells, the extracellular matrix (ECM) proteins, and the growth factors regulating the synthesis, degradation, and deposition of the ECM proteins will be discussed. Therapeutics with particular emphasis given to gene therapies and their effects on specific signaling pathways are described. Bleomycin (BM), a potent antineoplastic antibiotic increases TGF‐β1 transcription, TGF‐β1 gene expression, and TGF‐β protein. Like TGF‐β1, BM acts through the same distal promoter cis‐element of the COL1A1 gene causing increased COL1 synthesis and lung fibrosis. Lung fibroblasts exist as subpopulations with one subset predominately responding to fibrogenic stimuli which could be a specific cell therapeutic target for the onset and development of pulmonary fibrosis. J. Cell. Physiol. 211: 585–589, 2007.

Functional Effects of the Hypertrophic Cardiomyopathy R403Q Mutation Are Different in an α- or β-Myosin Heavy Chain Backbone

The R403Q mutation in the β-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly α-MHC as opposed to the β-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q α-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous α-MHC was largely replaced with transgenically encoded β-MHC. A His6 tag was cloned at the N terminus of the α-and β-MHC to facilitate protein isolation by Ni2+-chelating chromatography. Characterization of the R403Q α-MHC by the in vitro motility assay showed a 30-40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a β-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q α-S1 is ∼30% higher than for wild type, whereas the enzymatic activity for R403Q β-S1 is reduced by ∼10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.

Tissue Stretch Decreases Soluble TGF-β1 and Type-1 Procollagen in Mouse Subcutaneous Connective Tissue: Evidence From Ex Vivo and In Vivo Models

Transforming growth factor beta 1 (TGF‐β1) plays a key role in connective tissue remodeling, scarring, and fibrosis. The effects of mechanical forces on TGF‐β1 and collagen deposition are not well understood. We tested the hypothesis that brief (10 min) static tissue stretch attenuates TGF‐β1‐mediated new collagen deposition in response to injury. We used two different models: (1) an ex vivo model in which excised mouse subcutaneous tissue (N = 44 animals) was kept in organ culture for 4 days and either stretched (20% strain for 10 min 1 day after excision) or not stretched; culture media was assayed by ELISA for TGF‐β1; (2) an in vivo model in which mice (N = 22 animals) underwent unilateral subcutaneous microsurgical injury on the back, then were randomized to stretch (20–30% strain for 10 min twice a day for 7 days) or no stretch; subcutaneous tissues of the back were immunohistochemically stained for Type‐1 procollagen. In the ex vivo model, TGF‐β1 protein was lower in stretched versus non‐stretched tissue (repeated measures ANOVA, P < 0.01). In the in vivo model, microinjury resulted in a significant increase in Type‐1 procollagen in the absence of stretch (P < 0.001), but not in the presence of stretch (P = 0.21). Thus, brief tissue stretch attenuated the increase in both soluble TGF‐β1 (ex vivo) and Type‐1 procollagen (in vivo) following tissue injury. These results have potential relevance to the mechanisms of treatments applying brief mechanical stretch to tissues (e.g., physical therapy, respiratory therapy, mechanical ventilation, massage, yoga, acupuncture). J. Cell. Physiol. 214: 389–395, 2008.

GnRH Stimulates Expression of PACAP in the Pituitary Gonadotropes via Both the PKA and PKC Signaling Systems

Recent studies have demonstrated a clear role for pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of gonadotropin biosynthesis and secretion, both alone and in conjunction with GnRH. First defined as a hypothalamic releasing factor, PACAP subsequently has been identified in the gonadotrope subpopulation of the anterior pituitary gland, suggesting that PACAP may act as an autocrine-paracrine factor in this tissue. In initial studies, we determined that GnRH markedly stimulated endogenous PACAP mRNA levels and promoter-reporter activity in the mature gonadotrope cell line, LbetaT2. GnRH-stimulated rat PACAP promoter activity was blunted with deletion from position -915 to -402 and eliminated with further truncation to position -77 relative to the transcriptional start site. Site-directed mutagenesis demonstrated a functional requirement for a cAMP response element (CRE)-like site at position -205 and an activating protein-1 (AP-1)-like site at position -275, both of which bound CRE binding protein and AP-1 family members on EMSA. Treatment with pharmacological activators or inhibitors of second messenger signaling pathways implicated the protein kinase A, protein kinase C, and MAPK pathways in the GnRH response. In support of these in vitro data, we demonstrate that JunB binds to the rat PACAP gene promoter by chromatin immunoprecipitation assay and that small interfering RNA knockdown of JunB, cFos, and CRE binding protein factors blunts PACAP expression. In summary, these results further elucidate the complex functional interactions between PACAP and GnRH in the anterior pituitary. Specifically, these studies demonstrate that GnRH-stimulated PACAP gene expression is mediated via multiple signaling pathways acting on CRE/AP-1 sites in the proximal gene promoter. Because both PACAP and GnRH regulate gonadotropin biosynthesis and secretion, these results provide important insight into the critical fine tuning of gonadotrope function and, thereby, the maintenance of normal reproductive function.

Myosin heavy chain isoform expression in rat smooth muscle development

Smooth muscle myosin heavy chains (MHCs), the motor proteins that power smooth muscle contraction, are produced by alternative splicing from a single gene. The smooth muscle MHC gene is capable of producing four isoforms by utilizing alternative splice sites located at the regions encoding the carboxy terminus and the junction of the 25- and 50-kDa tryptic peptides. These four isoforms, SM1A, SM1B, SM2A, and SM2B, are a combination of one of two heavy chains containing different carboxy-terminal tails (1 or 2) without (A) or with (B) an additional motif in the myosin head. In the present study, using RNA analysis and isoform-specific antibodies, we demonstrate the expression patterns of MHC isoforms during development in rat smooth muscle tissues. RNase protection analysis indicates that the mRNAs for SMA and SMB isoforms, which differ by a 21-nucleotide insertion in the region encoding the S1 head region of the myosin molecule, are differentially expressed during development in a highly tissue-specific manner. Smooth muscle MHC transcripts are first detectable in developing rat smooth muscle tissues at 17 days of fetal development. The SMB mRNA is shown to be expressed in smooth muscle from fetal bladder, intestine, and stomach and from neonatal aorta; however, it is not expressed in cultured smooth muscle cells from rat aorta. The SMA mRNA is also present at all stages of development in the smooth muscles examined; however, it is much less abundant than SMB mRNA in most fetal smooth muscles. We show here that the SMB isoform, which contains a unique seven-amino acid insertion at the junction of the 25- and 50-kDa tryptic peptides, is present in conjunction with SM1 and SM2 tails on immunoblots of smooth muscle from stomach, intestine, bladder, and uterus and is expressed during development in a pattern distinct from that of the SM1 and SM2 tail isoforms.Smooth muscle myosin heavy chains (MHCs), the motor proteins that power smooth muscle contraction, are produced by alternative splicing from a single gene. The smooth muscle MHC gene is capable of producing four isoforms by utilizing alternative splice sites located at the regions encoding the carboxy terminus and the junction of the 25- and 50-kDa tryptic peptides. These four isoforms, SM1A, SM1B, SM2A, and SM2B, are a combination of one of two heavy chains containing different carboxy-terminal tails (1 or 2) without (A) or with (B) an additional motif in the myosin head. In the present study, using RNA analysis and isoform-specific antibodies, we demonstrate the expression patterns of MHC isoforms during development in rat smooth muscle tissues. RNase protection analysis indicates that the mRNAs for SMA and SMB isoforms, which differ by a 21-nucleotide insertion in the region encoding the S1 head region of the myosin molecule, are differentially expressed during development in a highly tissue-specific manner. Smooth muscle MHC transcripts are first detectable in developing rat smooth muscle tissues at 17 days of fetal development. The SMB mRNA is shown to be expressed in smooth muscle from fetal bladder, intestine, and stomach and from neonatal aorta; however, it is not expressed in cultured smooth muscle cells from rat aorta. The SMA mRNA is also present at all stages of development in the smooth muscles examined; however, it is much less abundant than SMB mRNA in most fetal smooth muscles. We show here that the SMB isoform, which contains a unique seven-amino acid insertion at the junction of the 25- and 50-kDa tryptic peptides, is present in conjunction with SM1 and SM2 tails on immunoblots of smooth muscle from stomach, intestine, bladder, and uterus and is expressed during development in a pattern distinct from that of the SM1 and SM2 tail isoforms.

Lung smooth muscle differentiation

The vascular and visceral smooth muscle tissues of the lung perform a number of tasks that are critical to pulmonary function. Smooth muscle function often is compromised as a result of lung disease. Though a great deal is known about regulation of smooth muscle cell replication and cell and tissue contractility, much less is understood regarding the phenotype of the contractile protein machinery of lung smooth muscle cells. This review focuses on the expression of cytoskeletal and contractile proteins of lung vascular and airway smooth muscle cells during development, in the adult and during vascular and airway remodeling. Emphasis is placed on the expression of the heavy chain of smooth muscle myosin, as well as the regulation of its gene. Important areas for future research are discussed.

Smooth muscle actin and myosin expression in cultured airway smooth muscle cells

In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express alpha- and gamma-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express α- and γ-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.

Microvessel precursor smooth muscle cells express head-inserted smooth muscle myosin heavy chain (SM-B) isoform in hyperoxic pulmonary hypertension.

Abstract The present study analyzes smooth muscle myosin heavy chain (SMMHC) expression as lung microvascular precursor smooth muscle cells (PSMCs), cells derived from fibroblasts and intermediate cells (immature SMCs), acquire a smooth muscle phenotype in anin vivo model of pulmonary hypertension (PH). Because of the unique contractile properties of the SMMHC isoform SM-B, we analyzed its expression in the microvessels (<100 μm diameter) and in larger vessels (100–700 μm) quantitatualy by the labeled [strept]avidin-biotin technique (day 1–28), and related this to cell phenotype by transmission microscopy and protein A-gold labeling (at day 28). Airway SMCs of the normal and hypertensive lung uniformly expressed SM-B whereas vascular SMC expression was heterogeneous. Thus, in some large arteries (and veins) SMCs contained cells expressing SM-B while in others all the cells were immunonegative. Microvascular cells expressing SM-B (arteries and veins) were rare in normal lung and numerous in PH, increasing as wall muscle developed in smaller segments with time. As in large vessels, some microvessels had immunopositive cells and others only negative ones. Ultrastructural analysis confirmed that the SMCs of bronchial vessels, and the septal SMCs adjoining alveolar ducts, contained dense filament arrays decorated with SM-B. While the PSMC processes of the normal lung contained sparse filaments decorated with SM-B, these cells expressed dense filament arrays in PH. Fibroblasts migrating to align around the microvessels also expressed SM-B but in the absence of a filament network. For the first time,we demonstrate in vivo that newly developed microvascular PSMCs express the SMMHC SM-B isoform in PH.

Tissue fibrosis and carcinogenesis: divergent or successive pathways dictate multiple molecular therapeutic targets for oligo decoy therapies.

The extracellular matrix (ECM) is composed of several families of macromolecular components: fibrous proteins such as collagens, type I collagen (COL1), type III collagen (COL3), fibronectin, elastin, and glycoconjugates such as proteoglycans and matrix glycoproteins. Their receptors on the cell membrane, most of which in the case of the ECM belong to the integrins, which are heterodimeric proteins composed of α and β chains. COL1 is the major fibrous collagen of bone, tendon, and skin; while COL3 is the more pliable collagen of organs like liver. Focus will not only be given to the regulation of synthesis of several fibrogenic parameters but also modulation of their degradation during growth factor‐induced tissue fibrosis and cancer development. Evidence will be provided that certain tissues, which undergo fibrosis, also become cancerous. Why does there exist a divergency between tissues, which undergo frank fibrosis as an endpoint, and those tissues that undergo fibrosis and subsequently are susceptible to carcinogenicity; resulting from the etiological factor(s) causing the initial injury? For example, why does a polyvinyl alcohol (PVA) sponge implant become encapsulated and filled with fibrous tissue then fibrosis tissue growth stops? Why does the subcutaneous injection of a fibrogenic growth factor cause a benign growth and incisional wounding results in fibrosis and ultimately scarring? There are many examples of tissues, which undergo fibrosis as a prerequisite to carcinogenesis. Is there a cause‐effect relationship? If you block tissue fibrosis in these precancerous tissues, would you block cancer formation? What are the molecular targets for blocking fibrosis and ultimately carcinogenesis? How can oligo decoys may be used to attenuate carcinogenesis and which oligo decoys specifically attenuate fibrogenesis as a prelude to carcinogenesis? What are other molecular targets for oligo decoy therapy in carcinogenesis? J. Cell. Biochem. 97: 1161–1174, 2006.