Biomedical subjects

Kenneth R Cutroneo

Publications and source records attributed to Kenneth R Cutroneo.

7 recordsLinked to original sources

PubMed2003

How is Type I procollagen synthesis regulated at the gene level during tissue fibrosis.

In response to tissue injury connective tissue synthesis occurs either normally or abnormally, which is mediated by transforming growth factor-beta (TGF-beta) and other growth factors. This article will be primarily concerned with the response of injured tissues at the gene level of Type I procollagen synthesis in response to TGF-beta. This leads to provisional repair, which in turn may lead to involution, remodeling, regeneration, and ultimately repair. Alternately, continuation of provisional repair may lead to fibrosis and ultimately scarring. Scarring of internal organs such as the liver and the lung leads to loss of function and ultimately death. In the case of scarring of skin, this is a cosmetic problem and can be rectified by surgery. Type I procollagen is synthesized by two genes, proalpha1 (Type I) and proalpha2 (Type I) collagen genes. This article will focus on DNA binding sites on these two genes, which regulate the transcription of the specific gene. This article will also define specific cell signaling pathways for the turning on of the proalpha1 and proalpha2 (Type I) collagen genes. This article will address several questions. First, what is the major cytokine acting extracellularly which stimulates the transcription of the proalpha1 and proalpha2 (Type I) collagen genes during tissue fibrosis? Secondly, how are the signals transmitted by the extracellular profibrotic cytokine TGF-beta from the cellular membrane to the nucleus for transcription of the proalpha1 (Type I) and proalpha2 (Type I) collagen genes? Thirdly, what signaling pathways cross-talk with the signaling pathways resulting in the expression of the Type I collagen genes? Fourthly, how does TGF-beta affect extracellular matrix homeostasis? Fifthly, what are the nuclear factors corresponding to the DNA elements required for the promotion of the proalpha1 (Type I) and proalpha2 (Type I) collagen genes? Finally, how are the proalpha1 (Type I) and proalpha2 (Type I) collagen genes coordinately regulated? Strategies will also be presented for reducing fibrosis, which is the result of overexpression of TGF-beta.

Animals↗

PubMed2003

TGF-beta1-induced Smad 3 binding to the Smad 7 gene: knockout of Smad 7 gene transcription by sense phosphorothioate oligos, autoregulation, and effect on TGF-beta1 secretion: bleomycin acts through TGF-beta1.

Bleomycin produces its fibrogenic effect, at least in part, by TGF-beta1 secretion. Treatment of IMR-90 human embryonic lung fibroblasts with bleomycin at 0.5 microg/ml results in a 1.6-fold increase of TGF-beta1 as determined by a specific ELISA assay for TGF-beta1 after acidification of the conditioned media. This elevation of TGF-beta1 secretion is furthermore enhanced in vivo by TGF-beta1 autoinduction of the TGF-beta1 gene. To demonstrate TGF-beta1 autoinduction, the fibroblasts were pretreated with 12.5 ng/ml TGF-beta1, washed extensively to remove any residual TGF-beta1, and then allowed to incubate for 24 h in AIM V synthetic serum-free media. The media when assayed using the ELISA assay contained a 1.6-fold increase of TGF-beta1. The distal promoter of the human TGF-beta1 gene contains a Smad 3 element (CAGGACA), which is homologous to the Smad 3 binding element motif (CAGA). The nuclear extracts of human embryonic lung fibroblasts treated for either 15 min or 24 h with TGF-beta1 did not demonstrate specificity of binding of a protein(s) to the homologous Smad 3 element as determined by cold wild-type oligodeoxynucleotide competition experiments. However, specific Smad 3 binding to the Smad 3 element (GTCTAGAC) found in proximal promoter of the Smad 7 gene was observed by cold oligo competition and supershift assays using a goat polyclonal Smad 3 antibody in the presence and absence of an N-terminal Smad 3 peptide. To determine the functionality of this Smad 3 binding to the Smad 3 element in the proximal promoter of the Smad 7 inhibitory gene to TGF-beta1 secretion, fibroblasts were transiently pretransfected with double-stranded phosphorothioate oligo "decoys" containing the Smad 7/Smad 3 element in the presence of plasmin to convert latent TGF-beta1 to active TGF-beta1. Under these conditions, which simulate the in vivo situation of 2.2-fold increase of total active TGF-beta1 was observed. Fibroblasts were also pretransfected with these double-stranded oligo "decoys," washed, then treated with TGF-beta1, washed and incubated in AIM V for an additional 24 h. In this latter experiment, a superinduction of TGF-beta1 secretion was observed. We propose that these oligo "decoys" bind Smad 3 preventing this initiation factor from binding to the Smad 7/Smad 3 element thereby decreasing the transcription of the Smad 7 gene. The decrease of the inhibitory Smad 7 would result in less binding of this Smad inhibitor to the Type I TGF-beta receptor and less antagonism of active TGF-beta1, more autoinduction of the TGF-beta1 gene, and more of the fibrogenic effects of TGF-beta1.

Bleomycin↗

PubMed2003

Gene therapy for tissue regeneration.

Tissue repair and regeneration are the normal biological responses of many different tissues in the body to injury. During the healing process, profound changes occur in cell composition and extracellular matrix (ECM) formation. Fibroblasts and equivalent reparative cells migrate to the wounded area and subsequently proliferate. These cells and reparative cells from the surrounding tissue are responsible for the rapid repair which results in tissue regeneration. Growth factors, one of which is transforming growth factor-beta (TGF-beta), stimulate fibroblasts and smooth muscle cells to proliferate and synthesize ECM proteins. This process of early repair provides a rapid way to restore new tissue and mechanical integrity. This early tissue repair process is normally followed by involution, which requires the production and activation of proteases, tissue maturation and remodeling, reorganization and finally regeneration. Alternately, failure to replace the critical components of the ECM, including elastin and basement membrane, results in abnormal regeneration of the epithelial cell layer. Although remodeling should occur during healing, provisional repair may be followed by excessive synthesis and deposition of collagen, which results in irreversible fibrosis and scarring. This excessive fibrosis which occurs in aberrant healing is at least in part mediated by persistent TGF-beta. Because of the central role of collagen in the wound healing process, the pharmacological control of collagen synthesis has been of paramount importance as a possible way to abrogate aberrant healing and prevent irreversible fibrosis. Fibrosis is an abnormal response to tissue injury.

Animals↗

PubMed2003

Sense oligonucleotide competition for gene promoter binding and activation.

Considerable evidence has ensued on the importance of growth factors during regeneration both for cell replication and for stimulation of reparative cells to synthesize and secrete extracellular matrix components. During the healing process if the growth factor concentration is too high because of over-expression, abnormal wound healing and tissue fibrosis will occur. The growth factor concentration at the wound site may be controlled by gene therapy and the titration of gene dosage. However, if there is a narrow window between the beneficial effects and adverse effects of gene therapy, oligonucleotide approaches may be used concurrently with gene therapy to control growth factor concentration(s) at the wound site. Antisense oligos offer a method to control the concentration of growth factors at the level of translation. A novel method using sense oligos to the proalpha1 (I) collagen gene to inhibit gene transcription and collagen synthesis has recently been reported. The exogenous modified oligodeoxynucleotide competes with the cis-element (i.e. the transforming growth factor-beta (TGF-beta) element) in the distal 5'-flanking region of the proalpha1 (I) collagen gene for the trans-acting factor (i.e. the TGF-beta activator protein complex), thereby down regulating promoter activity of the proalpha1 (I) collagen gene and inhibiting type I collagen synthesis. The oligonucleotide approaches, both antisense and sense therapies, may be used to regulate over-expression of growth factors and thereby either eliminate or lessen the potential adverse effects of gene therapy.

Animals↗

PubMed2002

Rational basis for oligodeoxynucleotides to inhibit collagen synthesis in lung fibroblasts and primary fibroblasts from liver granulomas of Schistosoma mansoni-infected mice.

Hepatocellular carcinoma is associated with liver fibrosis. Murine schistosomiasis infection offers a model to study hepatic fibrogenesis. Single-stranded phosphorothiate oligodeoxynucleotides containing the TGF-beta regulatory element have been shown to regulate the transcription of this gene and effectively inhibit collagen synthesis in primary fibroblasts isolated from schistosomiasis-induced hepatic granulomas. While the single-stranded oligos did not decrease collagen and non-collagen protein synthesis below control levels, their double-stranded modified and unmodified counterparts did. Competitive cold oligodeoxynucleotide gel mobility shift analysis using control fibroblast nuclear extract demonstrated that the single-stranded oligos diminished binding of the TGF-beta activator protein to the TGF-beta regulatory element while the double-stranded oligos totally inhibited this binding. TGF-beta element containing single-stranded phosphorothioate oligodeoxynucleotides and their double-stranded counterparts may be successful therapeutic agents to inhibit hepatic fibrogenesis and associated hepatocellular carcinoma.

Animals↗

PubMed2002

Differentiation-dependent induction of CYP1A1 in cultured rat small intestinal epithelial cells, colonocytes, and human colon carcinoma cells: basement membrane-mediated apoptosis.

Rat small intestinal epithelial cells and human colon adenocarcinoma cells cultured on Matrigel expressed the differentiation specific enzyme, sucrase-isomaltase, as determined by indirect immunofluorescence. Rat small intestinal epithelial cells, rat colonocytes, and human colon adenocarcinoma cells developed an altered morphology when cultured on Matrigel and became apoptotic within 24-48 h. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin caused a 2- and 5-fold induction, respectively, of ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene- or 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on plastic was not detected. 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment caused a 14-fold induction of transfected, rat CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment induced ethoxyresorufin-o-deethylase activity by 6- and 1.6-fold, respectively in rat colonocytes cultured on Matrigel. Induction of ethoxyresorufin-o-deethylase activity was not observed in rat colonocytes cultured on plastic. CYP1A1-promoter-luciferase activity was induced 3-fold by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat colonocytes cultured on Matrigel. Induction of CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells or rat colonocytes cultured on plastic was not observed. Ethoxyresorufin-o-deethylase activity in human colon adenocarcinoma cells, cultured on either plastic or Matrigel, was induced 7-fold by benzo[a]pyrene. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity was 2-fold greater in human colon adenocarcinoma cells cultured on Matrigel compared to cells cultured on plastic. Extracellular matrix-mediated differentiation and apoptosis of intestinal cells provide in vitro systems for study of the regulation of CYP1A1 expression, carcinogen activation in the gut and mechanism(s) of apoptosis of colon cancer cells.

Animals↗

PubMed2002

Relationship between glucocorticoid-mediated early decrease of protein synthesis and the steady state decreases of glucocorticoid receptor and TGF-beta activator protein.

The present studies were undertaken to better elucidate the mechanism(s) by which glucocorticoids inhibit the process of tissue repair. The aim was to determine the importance of the effect of glucocorticoids on decreasing the nuclear TGF-beta activator protein. The relationship amongst inhibition of noncollagen protein synthesis and the steady state levels of glucocorticoid receptor and the TGF-beta activator protein was examined. Both collagen and noncollagen synthesis were determined in skin fibroblast cell culture and in dermis. Fetal rat skin fibroblasts were treated for 24 h with dexamethsone. Noncollagen protein synthesis was decreased to approximately one-half that of collagen synthesis. Similar results were obtained in dermis in vivo. At 48 h, dexamethasone treatment resulted in practically no nuclear glucocorticoid receptor being noted and a 40-45% steady state decrease of the TGF-beta activator protein. We have recently reported that the TGF-beta activator protein exists as a protein complex with SP1 and NFKB (p 49). The present data indicate that although the marked decrease of the nuclear glucocorticoid receptor DNA binding following dexamethasone treatment is not comparable to the early 24 h decrease of noncollagen protein synthesis, the decrease of the TGF-beta activator protein complex binding to DNA is. The present studies indicate the importance of the effect of dexamethasone on the steady state level of the TGF-beta activator protein complex in the glucocorticoid-mediated process inhibition of tissue repair and the relationship of this decrease to the earlier inhibition of protein synthesis.

Animals↗