Arthur R. Strauch

Oxygen Sensing by Primary Cardiac Fibroblasts: A Key Role of p21Waf1/Cip1/Sdi1

Abstract— In mammalian organs under normoxic conditions, O2 concentration ranges from 12% to <0.5%, with O2 ≈14% in arterial blood and <10% in the myocardium. During mild hypoxia, myocardial O2 drops to ≈1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of Po2 results in perceived hyperoxia. We hypothesized that O2, even in marginal relative excess of the Po2 to which cardiac cells are adjusted, results in activation of specific signal transduction pathways that alter the phenotype and function of these cells. To test this hypothesis, cardiac fibroblasts (CFs) isolated from adult murine ventricle were cultured in 10% or 21% O2 (hyperoxia relative to the Po2 to which cells are adjusted in vivo) and were compared with those cultured in 3% O2 (mild hypoxia). Compared with cells cultured in 3% O2, cells that were cultured in 10% or 21% O2 demonstrated remarkable reversible G2/M arrest and a phenotype indicative of differentiation to myofibroblasts. These effects were independent of NADPH oxidase function. CFs exposed to high O2 exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p21, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming growth factor-&bgr;1, (2) lowered telomerase activity, and (3) activation of transforming growth factor-&bgr;1 and p38 mitogen-activated protein kinase. CFs deficient in p21 were resistant to such O2 sensitivity. This study raises the vital broad-based issue of controlling ambient O2 during the culture of primary cells isolated from organs.

Overexpression of Insulin-Like Growth Factor-Binding Protein-4 (IGFBP-4) in Smooth Muscle Cells of Transgenic Mice through a Smooth Muscle α-Actin-IGFBP-4 Fusion Gene Induces Smooth Muscle Hypoplasia

Insulin-like growth factor I (IGF-I) has been postulated to function as a smooth muscle cell (SMC) mitogen and to play a role in the pathogenesis of bladder hypertrophy, estrogen-induced uterine growth, and restenosis after arterial angioplasty. IGF-binding protein-4 (IGFBP-4) inhibits IGF-I action in vitro and is the most abundant IGFBP in the rodent arterial wall. To explore the function of this binding protein in vivo, transgenic mouse lines were developed harboring fusion genes consisting of a rat IGFBP-4 complementary DNA cloned downstream of either a −724 bp fragment of the mouse smooth muscle α-actin 5′-flanking region (SMP2-BP-4) or− 1074 bp, 63 bp of 5′-untranslated region, and 2.5 kb of intron 1 of smooth muscle α-actin (SMP8-BP-4). SMP2-BP-4 mice expressed low levels of the exogenous IGFBP-4 messenger RNA (mRNA), which was not specifically targeted to SMC-rich tissue environments, and were therefore not analyzed further. Six SMP8-BP-4 transgenic lines derived from separate founders were charact...

Sequence of cDNAs encoding components of vascular actin single-stranded DNA-binding factor 2 establish identity to Puralpha and Purbeta.

Transcriptional repression of the mouse vascular smooth muscle α-actin gene in fibroblasts and myoblasts is mediated, in part, by the interaction of two single-stranded DNA binding activities with opposite strands of an essential transcription enhancer factor-1 recognition element (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429–2436). One of these activities, previously designated vascular actin single-stranded DNA-binding factor 2 includes two distinct polypeptides (p44 and p46) which specifically interact with the purine-rich strand of both the enhancer and a related element in a protein coding exon of the gene (Kelm, R. J., Jr., Sun, S., Strauch, A. R., and Getz, M. J. (1996) J. Biol. Chem. 271, 24278–24285). Expression screening of a mouse lung cDNA library with a vascular actin single-stranded DNA-binding factor 2 recognition element has now resulted in the isolation of two distinct cDNA clones that encode p46 and p44. One of these proteins is identical to Purα, a retinoblastoma-binding protein previously implicated in both transcriptional activation and DNA replication. The other is a related family member, presumably Purβ. Comparative band shift and Southwestern blot analyses conducted with cellular p46, p44, and cloned Pur proteins synthesized in vitro and in vivo, establish identity of p46 with Purα and p44 with Purβ. This study implicates Purα and/or Purβ in the control of vascular smooth muscle α-actin gene transcription.

Morphometric analysis of neointimal formation in murine cardiac allografts.

BACKGROUND Transplant vascular sclerosis is expressed in transplanted human and murine hearts as a concentric intimal thickening. The purpose of this study was to characterize the location, distribution, and intensity of transplant vascular sclerosis in murine cardiac allografts using computerized morphometric analysis. METHODS Murine cardiac allograft recipients were treated with the immunosuppressant gallium nitrate to promote graft survival. The grafts were removed at 60 days after transplantation and histologically stained. The coronary arteries were analyzed for intimal thickening using a neointimal index (NI) derived with a computer imaging system. RESULTS A cross-section taken from the middle of a cardiac allograft showed four major coronary arteries, each with widely different NI values (65, 0, 92, and 0). The same four vessels in two other grafts also showed highly variable NI values, but different patterns of vessel involvement. Next, NI values were determined along the length of a single vessel from aorta to apex. This revealed variable, fluctuating intimal thickening along the length of the vessel. In general, arteries from the aortic versus apical regions of the grafted hearts expressed similar amounts of intimal thickening (analysis of variance, P=0.4826). Finally, a method was devised to quantitate intimal thickening from a sampling of three tissue cross-sections taken from the middle of each cardiac allograft. This value was statistically indistinguishable from values obtained by analysis of intimal thickening in multiple sections covering the entire heart (P=0.6734, 0.9021, and 0.1474). CONCLUSIONS Intimal thickening in the coronary arteries of murine cardiac allografts appears to be variable in terms of location, distribution, and intensity. This is true for different regions of the same vessel, different vessels in the same heart region, and the same vessels in different cardiac allografts.

Molecular interactions between single-stranded DNA-binding proteins associated with an essential MCAT element in the mouse smooth muscle alpha-actin promoter.

Transcriptional activity of the mouse vascular smooth muscle α-actin gene in fibroblasts is regulated, in part, by a 30-base pair asymmetric polypurine-polypyrimidine tract containing an essential MCAT enhancer motif. The double-stranded form of this sequence serves as a binding site for a transcription enhancer factor 1-related protein while the separated single strands interact with two distinct DNA binding activities termed VACssBF1 and 2 (Cogan, J. G., Sun, S., Stoflet, E. S., Schmidt, L. J., Getz, M. J., and Strauch, A. R. (1995) J. Biol. Chem. 270, 11310–11321; Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429–2936). VACssBF2 has been recently cloned and shown to consist of two closely related proteins, Purα and Purβ (Kelm, R. J., Elder, P. K., Strauch, A. R., and Getz, M. J. (1997)J. Biol. Chem. 272, 26727–26733). In this study, we demonstrate that Purα and Purβ interact with each other via highly specific protein-protein interactions and bind to the purine-rich strand of the MCAT enhancer in the form of both homo- and heteromeric complexes. Moreover, both Pur proteins interact with MSY1, a VACssBF1-like protein cloned by virtue of its affinity for the pyrimidine-rich strand of the enhancer. Interactions between Purα, Purβ, and MSY1 do not require the participation of DNA. Combinatorial interactions between these three single-stranded DNA-binding proteins may be important in regulating activity of the smooth muscle α-actin MCAT enhancer in fibroblasts.

Reprogramming of vascular smooth muscle α-actin gene expression as an early indicator of dysfunctional remodeling following heart transplant

OBJECTIVE Chronic rejection in cardiac allografts depletes vascular smooth muscle (VSM) alpha-actin from the coronary arterial smooth muscle bed while promoting its abnormal accumulation in cardiomyocytes and myofibroblasts. The objective was to determine if the newly discovered TEF1, MSY1, Puralpha and Purbeta VSM alpha-actin transcriptional reprogramming proteins (TRPs) were associated with development of chronic rejection histopathology in accepted murine cardiac allografts. METHODS A mouse heterotopic cardiac transplant model was employed using H2 locus-mismatched mouse strains (DBA/2 or FVB/N to C57BL/6). Recipients were immunosuppressed to promote long-term allograft acceptance and emergence of chronic rejection. Explanted grafts and isolated heart cells were evaluated for changes in the DNA-binding activity and subcellular distribution of VSM alpha-actin transcriptional regulatory proteins. RESULTS The DNA-binding activity of all four TRPs was high in the developing mouse ventricle, minimal in adult donor hearts and increased substantially within 30 days after transplantation. Immunohistologic analysis revealed nuclear localization of Purbeta and MSY1 particularly in fibrotic areas of the allograft myocardium demonstrating extravascular accumulation of VSM alpha-actin. Cardiomyocytes isolated from adult, non-transplanted mouse hearts not only exhibited less VSM alpha-actin expression and lower levels of TRPs compared to isolated cardiac fibroblasts or neonatal cardiomyocytes, but also contained a novel size variant of the MSY1 protein. CONCLUSION Accumulation of TRPs in cardiac allografts, particularly within the fibroblast-enriched myocardial interstitium, was consistent with their potential role in VSM alpha-actin gene reprogramming, fibrosis and dysfunctional remodeling following transplant. These nuclear protein markers could help stage peri-transplant cellular events that precede formation of graft-destructive fibrosis and coronary vasculopathy during chronic rejection.

Morphometric analysis of neointimal formation in murine cardiac grafts: III. Dissociation of intestitial fibrosis from neointimal formation.

BACKGROUND This study examined the relationship between transplant vascular sclerosis (TVS) and tissue fibrosis, features of chronic rejection that can develop rapidly in accepted heterotopic murine cardiac allografts. METHODS The rate of development of interstitial fibrosis or TVS development was determined by computerized analysis of tissue sections from DBA/2-->C57BL/6 heterotopic cardiac allografts after immunosuppression with gallium nitrate. RESULTS In accepted cardiac allografts, neointimal fibrosis developed by 30 days after transplant, whereas TVS was minimal by day 30, and maximal by day 60. Variable levels of fibrosis were found throughout the allografts. DBA/2-->DBA/2 cardiac isografts never displayed TVS in this time period, but displayed allograft-like fibrosis within 60 days of transplantation. CONCLUSIONS Interstitial fibrosis can be dissociated from the TVS development in this experimental model of chronic cardiac allograft rejection. Apparently, it is caused, at least in part, by alloantigen-independent factors other than TVS-related tissue ischemia.

Transforming Growth Factor β1-mediated Activation of the Smooth Muscle α-Actin Gene in Human Pulmonary Myofibroblasts Is Inhibited by Tumor Necrosis Factor-α via Mitogen-activated Protein Kinase Kinase 1-dependent Induction of the Egr-1 Transcriptional Repressor

Transforming growth factor (TGF) beta1 is a mediator of myofibroblast differentiation in healing wounds in which it activates transcription of the smooth muscle alpha-actin (SMalphaA) gene via dynamic interplay of nuclear activators and repressors. Targeting components of TGFbeta1 signaling may be an effective strategy for controlling myofibroblasts in chronic fibrotic diseases. We examined the ability of proinflammatory tumor necrosis factor (TNF)-alpha to antagonize TGFbeta1-mediated human pulmonary myofibroblast differentiation. TNF-alpha abrogated TGFbeta1-induced SMalphaA gene expression at the level of transcription without disrupting phosphorylation of regulatory Smads. Intact mitogen-activated protein kinase kinase (Mek)-extracellular signal-regulated kinase (Erk) kinase signaling was required for myofibroblast repression by TNF-alpha via induction of the early growth response factor-1 (Egr-1) DNA-binding protein. Egr-1 bound to the GC-rich SPUR activation element in the SMalphaA promoter and potently suppressed Smad3- and TGFbeta1-mediated transcription. Reduction in Smad binding to the SMalphaA promoter in TNF-alpha-treated myofibroblasts was accompanied by an increase in Egr-1 and YB-1 repressor binding, suggesting that the molecular mechanism underlying repression may involve competitive interplay between Egr-1, YB-1, and Smads. The ability of TNF-alpha to attenuate myofibroblast differentiation via modulation of a Mek1/Erk/Egr-1 regulatory axis may be useful in designing new therapeutic targets to offset destructive tissue remodeling in chronic fibrotic disease.

Structure/Function Analysis of Mouse Purβ, a Single-stranded DNA-binding Repressor of Vascular Smooth Muscle α-Actin Gene Transcription

Plasticity of smooth muscle α-actin gene expression in fibroblasts and vascular smooth muscle cells is mediated by opposing effects of transcriptional activators and repressors. Among these factors, three single-stranded DNA-binding proteins, Purα, Purβ, and MSY1, have been implicated as coregulators of a cryptic 5′-enhancer module. In this study, a molecular analysis of Purβ, the least well characterized member of this group, was conducted. Southwestern and Northwestern blotting of purified Purβ deletion mutants using smooth muscle α-actin-derived probes mapped the minimal single-stranded DNA/RNA-binding domain to a conserved region spanning amino acids 37–263. Quantitative binding assays indicated that the relative affinity and specificity of Purβ for single-stranded DNA were influenced by purine/pyrimidine content; by non-conserved regions outside amino acids 37–263; and by cell-derived proteins, specifically MSY1. When overexpressed in A7r5 vascular smooth muscle cells, Purβ (but not Purα) inhibited transcription of a smooth muscle-specific mouse α-actin promoter transgene. Structural domains required for Purβ repressor activity included the minimal DNA-binding region and a C-terminal domain required for stabilizing high affinity protein and nucleic acid interactions. Purβ inhibitory activity in transfected A7r5 cells was potentiated by MSY1, but antagonized by serum response factor, reinforcing the idea that interplay among activators and repressors may account for phenotypic changes in smooth muscle α-actin-expressing cell types.

Nucleoprotein Interactions Governing Cell Type-dependent Repression of the Mouse Smooth Muscle α-Actin Promoter by Single-stranded DNA-binding Proteins Purα and Purβ

Purα and Purβ are structurally related single-stranded DNA/RNA-binding proteins implicated in the control of cell growth and differentiation. The goal of this study was to determine whether Purα and Purβ function in a redundant, distinct, or collaborative manner to suppress smooth muscleα-actin gene expression in cell types relevant to wound repair and vascular remodeling. RNA interference-mediated loss-of-function analyses revealed that, although Purβ was the dominant repressor, the combined action of endogenous Purα and Purβ was necessary to fully repress the full-length smooth muscle α-actin promoter in cultured fibroblasts but to a lesser extent in vascular smooth muscle cells. The activity of a minimal core enhancer containing a truncated 5′ Pur repressor binding site was unaffected by knockdown of Purα and/or Purβ in fibroblasts. Conversely, gain-of-function studies indicated that Purα or Purβ could each independently repress core smooth muscle α-actin enhancer activity albeit in a cell type-dependent fashion. Biochemical analyses indicated that purified recombinant Purα and Purβ were essentially identical in terms of their binding affinity and specificity for GGN repeat-containing strands of several cis-elements comprising the core enhancer. However, Purα and Purβ exhibited more distinctive protein interaction profiles when evaluated for binding to enhancer-associated transcription factors in extracts from fibroblasts and vascular smooth muscle cells. These findings support the hypothesis that Purα and Purβ repress smooth muscle α-actin gene transcription by means of DNA strand-selective cis-element binding and cell type-dependent protein-protein interactions.