Airway smooth muscle inflammation is regulated by microRNA-145 in COPD.

Chronic obstructive pulmonary disease (COPD) is a common, highly debilitating disease of the airways, primarily caused by smoking. Chronic inflammation and structural remodelling are key pathological features of this disease, in part caused by the aberrant function of airway smooth muscle (ASM) cells under the regulation of transforming growth factor (TGF)-ß. miRNA are short, noncoding gene transcripts involved in the negative regulation of specific target genes, through their interactions with mRNA. Previous studies have proposed that mRNA-145 (miR-145) may interact with SMAD3, an important downstream signalling molecule of the TGF-ß pathway. TGF-ß was used to stimulate primary human ASM cells isolated from healthy nonsmokers, healthy smokers and COPD patients. This resulted in a TGF-ß-dependent increase in CXCL8 and IL-6 release, most notably in the cells from COPD patients. TGF-ß stimulation increased SMAD3 expression, only in cells from COPD patients, with a concurrent increased miR-145 expression. Regulation of miR-145 was found to be negatively controlled by pathways involving the MAP kinases, MEK-1/2 and p38 MAPK. Subsequent, overexpression of miR-145 (using synthetic mimics) in ASM cells from patients with COPD suppressed IL-6 and CXCL8 release, to levels comparable to the nonsmoker controls. Therefore, this study suggests that miR-145 negatively regulates pro-inflammatory cytokine release from ASM cells in COPD by targeting SMAD3.

Characterization of the dystrophin-glycoprotein complex in airway smooth muscle: role of δ-sarcoglycan in airway responsiveness.

The dystrophin-glycoprotein complex (DGC) is an integral part of caveolae microdomains, and its interaction with caveolin-1 is essential for the phenotype and functional properties of airway smooth muscle (ASM). The sarcoglycan complex provides stability to the dystroglycan complex, but its role in ASM contraction and lung physiology in not understood. We tested whether δ-sarcoglycan (δ-SG), through its interaction with the DGC, is a determinant of ASM contraction ex vivo and airway mechanics in vivo. We measured methacholine (MCh)-induced isometric contraction and airway mechanics in δ-SG KO and wild-type mice. Last, we performed immunoblotting and transmission electron microscopy to assess DGC protein expression and the ultrastructural features of tracheal smooth muscle. Our results reveal an age-dependent reduction in the MCh-induced tracheal isometric force and significant reduction in airway resistance at high concentrations of MCh (50.0 mg/mL) in δ-SG KO mice. The changes in contraction and lung function correlated with decreased caveolin-1 and ß-dystroglycan abundance, as well as an age-dependent loss of caveolae in the cell membrane of tracheal smooth muscle in δ-SG KO mice. Collectively, these results confirm and extend understanding of a functional role for the DGC in the contractile properties of ASM and demonstrate that this results in altered lung function in vivo.

Chronic exposure to perfluorinated compounds: Impact on airway hyperresponsiveness and inflammation.

Emerging epidemiological evidence reveals a link between lung disease and exposure to indoor pollutants such as perfluorinated compounds (PFCs). PFC exposure during critical developmental stages may increase asthma susceptibility. Thus, in a murine model, we tested the hypothesis that early life and continued exposure to two ubiquitous household PFCs, perfluorooctanoic acid (PFOA) and perflurooctanesulfonic acid (PFOS), can induce lung dysfunction that exacerbates allergen-induced airway hyperresponsiveness (AHR) and inflammation. Balb/c mice were exposed to PFOA or PFOS (4 mg/kg chow) from gestation day 2 to 12 wk of age by feeding pregnant and nursing dams, and weaned pups. Some pups were also sensitized and challenged with ovalbumin (OVA). We assessed lung function and inflammatory cell and cytokine expression in the lung and examined bronchial goblet cell number. PFOA, but not PFOS, without the OVA sensitization/challenge induced AHR concomitant with a 25-fold increase of lung macrophages. PFOA exposure did not affect OVA-induced lung inflammatory cell number. In contrast, PFOS exposure inhibited OVA-induced lung inflammation, decreasing total cell number in lung lavage by 68.7%. Interferon-γ mRNA in the lung was elevated in all PFC-exposed groups. Despite these effects, neither PFOA nor PFOS affected OVA-induced AHR. Our data do not reveal PFOA or PFOS exposure as a risk factor for more severe allergic asthma-like symptoms, but PFOA alone can induce airway inflammation and alter airway function.

Identification of functional CCAAT/enhancer-binding protein and Ets protein binding sites in the human chorionic somatomammotropin enhancer sequences.

The human chorionic somatomammotropin (CS) A and B genes (listed as CSH1 and CSH2 in the HUGO database) are highly expressed in placenta. A 241 bp potent enhancer, nucleotides (nts) 1-241, located at the 3' end of the CS-B gene (CS-Benh) stimulates promoter activity specifically in placental trophoblast cells in vitro. Strong activity is exerted by a 23 bp element within the CS-Benh (nts 117-139), shown to interact with transcription enhancer factor (TEF) members of the transcription enhancer activator (TEA) DNA-binding domain-containing family. An identical TEF element is present in the homologous (97.5%) CS-Aenh; however, a few nucleotide differences suppress its activity. Previously, we identified regulatory sequences distinct from the TEF element within an 80 bp modulatory domain (nts 1-80) in the CS-Benh. Using structural and functional assays we now show that CCAAT/enhancer-binding protein (C/EBP) binding sites exist in the 80 bp modulatory domains of both enhancers, and an Elk-1 binding site exists in the modulatory domain of the CS-Aenh. C/EBPα or C/EBPß strongly repressed CSp.CAT activity but stimulated CSp.CAT.CS-Benh activity. In contrast, the equivalent CS-A enhancer sequences were unable to relieve promoter repression. Elk-1 overexpression also resulted in differential effects on the CS-Aenh versus CS-Benh. Finally, we provide evidence for the association of C/EBPß with the CS-A and CS-B genes in human placental chromatin, including differential involvement of C/EBPß with the CS-Aenh versus the CS-Benh, and therefore consistent with the notion that these are regions of regulatory significance in vivo. We conclude that members of the C/EBP and Ets families can differentially modulate CS-Benh and CS-Aenh activity.

FGF-16 is a target for adrenergic stimulation through NF-kappaB activation in postnatal cardiac cells and adult mouse heart.

AIMS: The fibroblast growth factor (FGF) family plays an important role in cardiac growth and development. However, only FGF-16 RNA levels are reported to increase during the perinatal period and to be expressed preferentially in the myocardium, suggesting control at the transcriptional level and a role for FGF-16 in the postnatal heart. Beyond the identification of two TATA-like elements (TATA1 and TATA2) in the mouse FGF-16 promoter region and the preferential cardiac activity of TATA2, there is no report of Fgf-16 gene regulation. Assessment of promoter sequences, however, reveals putative nuclear factor-kappaB (NF-kappaB) elements, suggesting that Fgf-16 is regulated via NF-kappaB activation and thereby implicated in a number of cardiac events. Thus, the Fgf-16 gene was investigated as a target for NF-kappaB activation in cardiac cells. METHODS AND RESULTS: Assessments of Fgf-16 promoter activity were made using truncated and transfected hybrid genes with NF-kappaB inhibitors and/or beta-adrenergic stimulation via isoproterenol (IsP) treatment (a known NF-kappaB activator) in culture, and on endogenous mouse and human Fgf-16 genes in situ. The mouse Fgf-16 promoter region was stimulated in response to IsP treatment, but this response was lost with NF-kappaB inhibitor pretreatment. Deletion analysis revealed IsP responsiveness linked to sequences between TATA2 and TATA1 and, more specifically, a NF-kappaB element upstream and adjacent to TATA1 that associates with NF-kappaB p50/p65 subunits in chromatin. Finally, TATA1 and the proximal NF-kappaB element are conserved in the human genome and responsive to IsP. CONCLUSION: The mouse and human Fgf-16 gene is a target for NF-kappaB activation in the postnatal heart.

FGF-16 is required for embryonic heart development.

Fibroblast growth factor 16 (FGF-16) expression has previously been detected in mouse heart at mid-gestation in the endocardium and epicardium, suggesting a role in embryonic heart development. More specifically, exogenously applied FGF-16 has been shown to stimulate growth of embryonic myocardial cells in tissue explants. We have generated mice lacking FGF-16 by targeting the Fgf16 locus on the X chromosome. Elimination of Fgf16 expression resulted in embryonic death as early as day 11.5 (E11.5). External abnormalities, including hemorrhage in the heart and ventral body region as well as facial defects, began to appear in null embryos from E11.5. Morphological analysis of FGF-16 null hearts revealed cardiac defects including chamber dilation, thinning of the atrial and ventricular walls, and poor trabeculation, which were visible at E10.5 and more pronounced at E11.5. These findings indicate FGF-16 is required for embryonic heart development in mid-gestation through its positive effect on myocardial growth.

FGF-16 is released from neonatal cardiac myocytes and alters growth-related signaling: a possible role in postnatal development.

FGF-16 has been reported to be preferentially expressed in the adult rat heart. We have investigated the expression of FGF-16 in the perinatal and postnatal heart and its functional significance in neonatal rat cardiac myocytes. FGF-16 mRNA accumulation was observed by quantitative RT-PCR between neonatal days 1 and 7, with this increased expression persisting into adulthood. FGF-2 has been shown to increase neonatal rat cardiac myocyte proliferative potential via PKC activation. Gene array analysis revealed that FGF-16 inhibited the upregulation by FGF-2 of cell cycle promoting genes including cyclin F and Ki67. Furthermore, the CDK4/6 inhibitor gene Arf/INK4A was upregulated with the combination of FGF-16 and FGF-2 but not with either factor on its own. The effect on Ki67 was validated by protein immunodetection, which also showed that FGF-16 significantly decreased FGF-2-induced Ki67 labeling of cardiac myocytes, although it alone had no effect on Ki67 labeling. Inhibition of p38 MAPK potentiated cardiac myocyte proliferation induced by FGF-2 but did not alter the inhibitory action of FGF-16. Receptor binding assay showed that FGF-16 can compete with FGF-2 for binding sites including FGF receptor 1. FGF-16 had no effect on activated p38, ERK1/2, or JNK/SAPK after FGF-2 treatment. However, FGF-16 inhibited PKC-alpha and PKC-epsilon activation induced by FGF-2 and, importantly, IGF-1. Collectively, these data suggest that expression and release of FGF-16 in the neonatal myocardium interfere with cardiac myocyte proliferative potential by altering the local signaling environment via modulation of PKC activation and cell cycle-related gene expression.

Fibroblast growth factor-2 and cardioprotection.

Boosting myocardial resistance to acute as well as chronic ischemic damage would ameliorate the detrimental effects of numerous cardiac pathologies and reduce the probability of transition to heart failure. Experimental cardiology has pointed to ischemic and pharmacological pre- as well as post-conditioning as potent acute cardioprotective manipulations. Additional exciting experimental strategies include the induction of true regenerative and/or angiogenic responses to the damaged heart, resulting in sustained structural and functional beneficial effects. Fibroblast growth factor-2 (FGF-2), an endogenous multifunctional protein with strong affinity for the extracellular matrix and basal lamina and well-documented paracrine, autocrine and intracellular modes of action, has been shown over the years to exert acute and direct pro-survival effects, irrespectively of whether it is administered before, during or after an ischemic insult to the heart. FGF-2 is also a potent angiogenic protein and a crucial agent for the proliferation, expansion, and survival of several cell types including those with stem cell properties. Human clinical trials have pointed to a good safety record for this protein. In this review, we will present a case for the low molecular weight isoform of fibroblast growth factor-2 (lo-FGF-2) as a very promising therapeutic agent to achieve powerful acute as well as sustained benefits for the heart, due to its cytoprotective and regenerative properties.

Regulation of the human growth hormone gene family: possible role for Pit-1 in early stages of pituitary-specific expression and repression.

The somatic cells of a multicellular organism contain an identical complement of genes that need to be expressed specifically and appropriately to allow the normal development and functions associated with an organism. In the eukaryotic cell nucleus, genes are packaged with nucleoprotein histones into chromatin. The human growth hormone (GH)/chorionic somatomammotropin (CS) gene family offers an excellent model to study the relationship between chromatin structure and transcription factor binding in terms of tissue-specific gene expression. The GH/CS gene family consists of five genes (GH-N, GH-V, CS-A, CS-B and CS-L), contained in a single locus on chromosome 17. Although they share approximately 94% sequence similarity, GH-N expression is restricted to pituitary somatotropes while the four placental GH/CS genes are expressed in the villus syncytiotrophoblast. Appropriate expression in vivo is dependent on remote sequences found 14-32 kb upstream of GH-N in the loci of adjacent genes, and these sequences are characterized by five (I-V) nuclease-hypersensitive sites (HS). Pituitary-specific factor Pit-1 binds at HS I/II and plays an essential role in chromatin remodeling and GH-N expression; however, the processes that lead to HS I/II accessibility are unknown. We discuss the possibility that Pit-1-driven remodeling at HS III may precede that at HS I/II in the pituitary. Also, in pituitary chromatin, all five GH/CS genes share similar nuclease sensitivity, suggesting that the conformation of the placental genes is not inhibitory to transcription. Given that the promoters of both GH-N and the placental GH/CS genes contain Pit-1-binding sites, possible mechanisms to restrict placenta GH/CS promoter activity in the pituitary are discussed, including active repression via P sequences located upstream of each of the placental GH/CS genes. Positively or negatively influencing those components known to be important for pituitary transcription may link epigenetic events to key transcription factors in the overall picture of tissue-specific control of gene expression.

Hepatocyte nuclear factor-3alpha binding at P sequences of the human growth hormone locus is associated with pituitary repressor function.

The human GH family consists of five genes, including the placental chorionic somatomammotropins (CS), within a single locus on chromosome 17. Based on nuclease sensitivity, the entire GH/CS locus is accessible in pituitary chromatin, yet only GH-N is expressed. Previously, we reported a P sequence element (263P) capable of repressing placental CS-A promoter activity in transfected pituitary (GC) cells, and our data indicated a possible role for nuclear factor-1 (NF-1) and regulatory factor X1 in this repression. In this study we show the formation of two independent pituitary complexes in vitro: a repressor complex containing NF-1 and a nonfunctional complex containing regulatory factor X1. In vitro repressor function is stabilized by the presence of P sequence element C (PSE-C), downstream of the previously characterized PSE-A and PSE-B. Repressor function is also dependent on an intact Pit-1 binding site in the CS-A promoter. EMSAs with PSE-C reveal binding of the hepatocyte nuclear factor-3/forkhead (HNF-3/fkh) family of transcription factors in rat pituitary GC cells. This observation is extended to human pituitary tissue, where HNF-3alpha's association with P sequences is confirmed by chromatin immunoprecipitation. Furthermore, protein-protein interactions between HNF-3alpha and NF-1 family members are demonstrated. These results identify HNF-3alpha as an additional member of the pituitary P sequence regulatory complex, implicating it in tissue-specific expression of the human GH/CS family.

Transcriptional regulation of FGF-2 gene expression in cardiac myocytes.

OBJECTIVE: Fibroblast growth factor-2 (FGF-2) exerts its cardioprotective effect through cell surface receptor signaling and may play a role in the normal maintenance of a healthy myocardium. One mechanism of FGF-2 release from contracting cardiomyocytes is through transient sarcolemmal disruption, with accumulation in the extracellular matrix. Continuous FGF-2 release would require a link to synthesis and, thus, we examined regulation of FGF-2 promoter activity in cardiomyocytes as a potential target for optimizing cardioprotection. METHODS AND RESULTS: To investigate autoregulation, neonatal rat cardiomyocytes, (NRCM), were transfected with approximately 1 or 0.1 kb of rat FGF-2 promoter sequences linked to luciferase, -1058FGF-2p.luc and -110FGF-2p.luc, and treated with or without FGF-2. FGF-2 promoter activity was significantly increased approximately 2.5-fold with both genes. The proximal promoter region of rat FGF-2 contains putative binding sites for the early growth response-1 (Egr-1) and stimulating protein 1 (Sp1) transcription factors. Overexpression of Egr-1 and Sp1 increased -1058FGF-2p.luc expression by 4.4- and 8.7-fold, respectively. Mutation of Egr-1 and overlapping Sp1 sites did not blunt the response of -110FGF-2p.luc to FGF-2 treatment but did significantly reduce basal promoter activity. Transgenic mice expressing -1058FGF-2p.luc were treated with isoproterenol (IsP) to increase heart rate and endogenous FGF-2 release. FGF-2 promoter activity was stimulated significantly at 6 h, and increases in both FGF-2 and its receptor mRNA levels were also detected. In contrast, no effect of IsP was seen on -1058FGF-2p.luc or -110FGF-2p.luc in transfected NRCMs. CONCLUSIONS: FGF-2 released from cardiomyocytes may act to regulate its own synthesis at the transcriptional level. The mechanism does not appear to require an intact Egr-1 site in the proximal promoter region. This may, however, reflect redundancy in the control of FGF-2 promoter activity as our data support a stimulatory role for Egr-1 and Sp1.

Beyond angiogenesis: the cardioprotective potential of fibroblast growth factor-2.

In the field of cardiovascular research, a number of independent approaches have been explored to protect the heart from acute and chronic ischemic damage. Fibroblast growth factor-2 (FGF-2) recently has received considerable attention with respect to its angiogenic potential. While therapeutic angiogenesis may serve to salvage chronically ischemic myocardium, more acute treatments are in demand to increase cardiac resistance to injury (preconditioning) and to guard against secondary injury after an acute ischemic insult. Here, we look beyond the angiogenic potential of FGF-2 and examine its acute cardioprotective activity as demonstrated under experimental conditions, both as an agent of a preconditioning-like response and for secondary injury prevention at the time of reperfusion. Factors to consider in moving to the clinical setting will be discussed, including issues of dosage, treatment duration, and routes of administration. Finally, issues of safety and clinical trial design will be considered. The prospect of such a multipotent growth factor having clinical usefulness opens the door to effective treatment of both acute and chronic ischemic heart disease, something well worth the attention of the cardiovascular community.

Biological activities of fibroblast growth factor-2 in the adult myocardium.

Fibroblast growth factor-2 (FGF-2) is a potent regulator of many cellular functions and phenomena, including cell proliferation, differentiation, survival, adhesion, migration, motility and apoptosis, and processes such as limb formation, wound healing, tumorigenesis, angiogenesis, vasculogenesis and blood vessel remodeling. In the adult myocardium, FGF-2 is expressed by various cell types, including cardiomyocytes, fibroblasts and smooth muscle cells. The biological effects of FGF-2 in the myocardium are mediated by the high-affinity tyrosine kinase receptor FGFR-1, the major FGF receptor in the heart. Here, we give an overview of current insights into the multiple roles of FGF-2 in the myocardium, as they pertain to two basic phenomena: ischemia-reperfusion injury and cardiac hypertrophy. The first category includes roles for FGF-2 in cardioprotection, the inflammatory response, angiogenesis and vascular remodeling, while the second includes myocyte hypertrophy, fibrosis, and gap junction functioning (conduction). Given the strong evidence for FGF-2 as both a cardioprotective and angiogenic agent, the therapeutic potential of FGF-2 in the ischemic myocardium is discussed.