Impaired fibrinolytic activity within the lung is a common manifestation of acute and chronic inflammatory lung diseases. Because the fibrinolytic system is active during repair processes that restore injured tissues to normal, reduced fibrinolytic activity may contribute to the subsequent development of pulmonary fibrosis. To examine the relationship between the fibrinolytic system and pulmonary fibrosis, lung inflammation was induced by bleomycin in transgenic mice that either overexpressed or were completely deficient in murine plasminogen activator inhibitor-1 (PAI-1). 2 wk after 0.075 U of bleomycin, the lungs of transgenic mice overexpressing PAI-1 contained significantly more hydroxyproline (118 Ϯ 8 g) than littermate controls (70.5 Ϯ 8 g, P Ͻ 0.005). 3 wk after administration of a higher dose of bleomycin (0.15 U), the lung hydroxyproline content of mice completely deficient in PAI-1 (49 Ϯ 8 g) was not significantly different ( P ϭ 0.63) than that of control animals receiving saline (37 Ϯ 1 g), while hydroxyproline content was significantly increased in heterozygote (77 Ϯ 12 g, P ϭ 0.06) and wild-type (124 Ϯ 19 g, P Ͻ 0.001) littermates. These data demonstrate a direct correlation between the genetically determined level of PAI-1 expression and the extent of collagen accumulation that follows inflammatory lung injury. These results strongly support the hypothesis that alterations in fibrinolytic activity influence the extent of pulmonary fibrosis that occurs after inflammatory injury.

The slow/cardiac troponin C (cTnC) gene has been used as a model system for defining the molecular mechanisms that regulate cardiac and skeletal muscle-specific gene expression during mammalian development. cTnC is expressed continuously in both embryonic and adult cardiac myocytes but is expressed only transiently in embryonic fast skeletal myotubes. We have reported previously that cTnC gene expression in skeletal myotubes is controlled by a developmentally regulated, skeletal muscle-specific transcriptional enhancer located within the first intron of the gene (bp 997 to 1141). In this report, we show that cTnC gene expression in cardiac myocytes both in vitro and in vivo is regulated by a distinct and independent transcriptional promoter and enhancer located within the immediate 5' flanking region of the gene (bp -124 to +32). DNase I footprint and electrophoretic mobility shift assay analyses demonstrated that this cardiac-specific promoter/enhancer contains five nuclear protein binding sites (designated CEF1, CEF-2, and CPF1-3), four of which bind novel cardiac-specific nuclear protein complexes. Functional analysis of the cardiac-specific cTnC enhancer revealed that mutation of either the CEF-1 or CEF-2 nuclear protein binding site abolished the activity of the cTnC enhancer in cardiac myocytes. Taken together, these results define a novel mechanism for developmentally regulating a single gene in multiple muscle cell lineages. In addition, they identify previously undefined cardiac-specific transcriptional regulatory motifs and trans-acting factors.

To address trafficking of transplanted marrow cells immediately after intravenous infusion, we examined the early fate of infused non‐adherent, low‐density donor bone marrow cells in a syngeneic mouse model. The presence of infused donor cells, marked with indium‐111 oxine (111In), with the fluorescent dye PKH26, or by a detectable transgene marker, was evaluated at 3–48 h in a variety of tissues, including peripheral blood. All three cell‐marking methods indicated a rapid (< 4 h) influx of cells into the bone marrow, liver, spleen, muscle and other tissues. Moreover, these tissues remained positive for the 48 h observation period. Interestingly, analysis of PKH26‐positive cells in non‐myeloablated animals demonstrated that approximately 17% of infused donor marrow cells localized to the marrow space within 15 h, whereas a smaller proportion of donor cells (~ 1–2%) localized to the marrow in recipients preconditioned by irradiation. In an effort to enrich for cells that specifically home to the bone marrow, PKH26‐labelled donor marrow cells were recovered from the first host and infused into a secondary recipient. Although this was a phenotypically undefined population of cells, no increase was observed in the relative fraction of PKH26‐labelled cells returning or ‘homing’ to the marrow of the second recipient. Taken together, these data suggest both that marrow engraftment may be mediated by non‐specific ‘seeding’ rather than a specific homing signal, and that efficient targeting of transplanted cells to the marrow is a complex multifaceted process.

The slow/cardiac troponin C (cTnC) gene is expressed in three distinct striated muscle lineages: cardiac myocytes, embryonic fast skeletal myotubes, and adult slow skeletal myocytes. We have reported previously that cTnC gene expression in cardiac muscle is regulated by a cardiac-specific promoter/enhancer located in the 5' flanking region of the gene (bp -124 to +1). In this report, we demonstrate that the cTnC gene contains a second distinct and independent transcriptional enhancer which is located in the first intron. This second enhancer is skeletal myotube specific and is developmentally up-regulated during the differentiation of myoblasts to myotubes. This enhancer contains three functionally important nuclear protein binding sites: a CACCC box, a MEF-2 binding site, and a previously undescribed nuclear protein binding site, designated MEF-3, which is also present in a large number of skeletal muscle-specific transcriptional enhancers. Unlike most skeletal muscle-specific transcriptional regulatory elements, the cTnC enhancer does not contain a consensus binding site (CANNTG) The expression of many muscle-specific proteins is developmentally regulated at the level of transcription (7,34,39,66,85). Thus, one approach to understanding the molecular basis of mammalian myogenesis is to elucidate the transcriptional mechanisms that regulate the expression of muscle-specific genes. The identification and characterization of the basic helix-loop-helix (bHLH) family of muscle-determining transcription factors, including MyoD, myogenin, myf-5, and MRF4/herculin/myf-6, has added significantly to our understanding of skeletal myogenesis (1,3,10,17,49,61,80). Expression of each of these transcription factors appears to be sufficient to activate the skeletal muscle phenotype in many types of cultured cells (77), and hexanucleotide binding sites for these factors (CANNTG), termed E boxes, have been identified in most but not all skeletal muscle-specific transcriptional regulatory elements studied to date (reviewed in reference 71). bHLH proteins each contain a basic domain which is required for DNA binding and an HLH region which is involved in the formation of homo-and heterodimers (6,9,74). The basic domain of each myogenic bHLH family member contains conserved alanine and threonine residues that distinguish them from the nonmyogenic bHLH transcription factors (5, 9).

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