Expression of tissue transglutaminase in the developing chicken limb is associated both with apoptosis and endochondral ossification.

The cross-linking enzyme tissue transglutaminase (tTG) participates in a variety of cellular functions. To assess its contribution to extracellular and intracellular processes during development we cloned the cDNA for chicken heart tissue transglutaminase and localized the sites of transglutaminase expression by in situ hybridization and immunohistochemistry. Compared with the chicken red blood cell transglutaminase cDNA, the heart cDNA encodes a transglutaminase with an amino-terminal truncation. The truncated enzyme retains full catalytic activity and is GTP-inhibitable. Tissue transglutaminase expression was observed in developmentally transient structures in embryonic chicken limb at day 7.5 of incubation suggesting that its expression is dynamically regulated during limb morphogenesis. The major morphogenetic events of the limb associated with transglutaminase expression were cartilage maturation during skeletal development, interdigital apoptosis, and differentiation of skeletal muscle. Maturation of the cartilage during endochondral ossification was characterized by intra- and extracellular transglutaminase accumulation in the zone of hypertrophic chondrocytes. Only intracellular enzyme could be detected in mesenchymal cells of the prospective joints, in apoptotic cells of the interdigital web, and in skeletal muscle myoblasts. An apparently constitutive expression of tissue transglutaminase was found in vascular endothelial cells corresponding to the adult expression pattern. The dynamic pattern of transglutaminase expression during morphogenesis suggests that tissue remodeling is a major trigger for transglutaminase induction.

Activators of the nuclear receptor PPARgamma enhance colon polyp formation.

A high-fat diet increases the risk of colon, breast and prostate cancer. The molecular mechanism by which dietary lipids promote tumorigenesis is unknown. Their effects may be mediated at least in part by the peroxisome proliferator-activated receptors (PPARs). These ligand-activated nuclear receptors modulate gene expression in response to fatty acids, lipid-derived metabolites and antidiabetic drugs. To explore the role of the PPARs in diet-induced carcinogenesis, we treated mice predisposed to intestinal neoplasia with a synthetic PPARgamma ligand. Reflecting the pattern of expression of PPARgamma in the gastrointestinal tract, treated mice developed a considerably greater number of polyps in the colon but not in the small intestine, indicating that PPARgamma activation may provide a molecular link between a high-fat diet and increased risk of colorectal cancer.

PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL.

The formation of foam cells from macrophages in the arterial wall is characterized by dramatic changes in lipid metabolism, including increased expression of scavenger receptors and the uptake of oxidized low-density lipoprotein (oxLDL). We demonstrate here that the nuclear receptor PPARgamma is induced in human monocytes following exposure to oxLDL and is expressed at high levels in the foam cells of atherosclerotic lesions. Ligand activation of the PPARgamma:RXRalpha heterodimer in myelomonocytic cell lines induces changes characteristic of monocytic differentiation and promotes uptake of oxLDL through transcriptional induction of the scavenger receptor CD36. These results reveal a novel signaling pathway controlling differentiation and lipid metabolism in monocytic cells, and suggest that endogenous PPARgamma ligands may be important regulators of gene expression during atherogenesis.

Retinoid-induced apoptosis in normal and neoplastic tissues.

Vitamin A and its derivatives (collectively referred to as retinoids) are required for many fundamental life processes, including vision, reproduction, metabolism, cellular differentiation, hematopoesis, bone development, and pattern formation during embryogenesis. There is also considerable evidence to suggest that natural and synthetic retinoids have therapeutical effects due to their antiproliferative and apoptosis-inducing effects in human diseases such as cancer. Therefore it is not surprising that a significant amount of research was dedicated to probe the molecular and cellular mechanisms of retinoid action during the past decade. One of the cellular mechanisms retinoids have been implicated in is the initiation and modulation of apoptosis in normal development and disease. This review provides a brief overview of the molecular basis of retinoid signaling, and focuses on the retinoid-regulation of apoptotic cell death and gene expression during normal development and in pathological conditions in vivo and in various tumor cell lines in vitro.

The promoter of the mouse tissue transglutaminase gene directs tissue-specific, retinoid-regulated and apoptosis-linked expression.

Tissue transglutaminase is a multifunctional enzyme that accumulates to high levels in cells undergoing apoptosis. Retinoids act as an acute and direct regulator of tissue transglutaminase gene transcription. The studies reported here were carried out to elucidate the molecular mechanisms involved in the regulation of tissue transglutaminase expression. We have isolated and characterized the mouse tissue transglutaminase gene promoter and 3.8 kb of 5'-flanking DNA. A large fragment of the promoter that includes both the core promoter and 3.8 kb of 5'-flanking DNA shows retinoid-dependent transcriptional activity when stably transfected into HeLa cells. In these stably transfected HeLa cells both the endogenous tissue transglutaminase gene and transfected mouse tissue transglutaminase promoter are activated by all-trans retinoic acid and by retinoic acid receptor (RAR)-specific and retinoid X receptor (RXR)-specific retinoids. In embryos made transgenic with a transglutaminase promoter-beta-galactosidase reporter gene, the transgene shows specific patterns of expression during limb development. The transglutaminase transgene is expressed in cartilage, the cells of the apical ectodermal ridge, and in regions of apoptotic cell death of the interdigital mesenchyme. It appears that cis-acting elements responsible for the complex retinoid regulation, tissue- and apoptosis-specific expression are embedded within the proximal 3.8 kb of DNA flanking the 5'-end of the mouse tissue transglutaminase gene.

Retinoid-induced epiphyseal plate closure in guinea pigs.

Vitamin A and its derivatives (retinoids) have been known to cause premature epiphyseal closure in humans as an unwanted side effect of chronic treatment. The purpose of the present study was to determine if guinea pigs could serve as an animal model of retinoid-induced epiphyseal plate closure, and to utilize this model to study the mechanism. Weanling male Hartley guinea pigs were treated ip via osmotic pump for up to 14 days with vehicle or 0.50 to 5.5 mg/kg/day of the retinoic acid receptor (RAR)-selective agonist AGN 190121. Histopathological examination of the proximal tibia of AGN 190121-treated guinea pigs revealed a dose-dependent disruption of the epiphyseal plate. The natural retinoids all-trans-retinoic acid and 13-cis-retinoic acid also induced epiphyseal plate closure in guinea pigs when administered by ip injection for 10 days. Prominent histological features of retinoid-induced epiphyseal closure included the loss of basophilic staining in the extracellular matrix of epiphyseal plate chondrocytes and the invasion of the epiphyseal plate by osteoclasts. To determine if the epiphyseal closure detected histologically was reversible, guinea pigs were treated for 6 days with the RAR-selective agonist (E)-4[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propen- 1-y1]benzoic acid (TTNPB) or vehicle, and groups of guinea pigs were euthanized on Day 7 or 57. TTNPB but not vehicle treatment caused histological evidence of epiphyseal closure at both time points, and significant bone elongation between Day 7 and Day 57 was detected only in vehicle-treated animals. Epiphyseal closure and other toxic effects of TTNPB were blocked by cotreatment of guinea pigs with a fivefold molar excess of AGN 193109, an RAR antagonist. Taken together, these data demonstrate the utility of the guinea pig as an animal model of retinoid-induced epiphyseal closure and suggest that RAR activation is necessary and sufficient for this activity.

Retinoid-regulated expression of BCL-2 and tissue transglutaminase during the differentiation and apoptosis of human myeloid leukemia (HL-60) cells.

Retinoids induce terminal differentiation and subsequent apoptosis in the human myeloid leukemia (HL-60) cell line. We have previously shown that in HL-60 cells, ligand activation of retinoic acid receptors (RARs) is sufficient to induce differentiation but ligand activation of retinoid X receptors (RXRs) is necessary for the retinoid-induced apoptosis of these cells. In the present studies we have characterized the effect of retinoids on the expression of two apoptosis-linked gene products, BCL-2 and tissue transglutaminase. BCL-2 is a membrane-associated protein whose expression has been linked to the suppression of apoptosis in many cells. Tissue transglutaminase is a protein cross-linking enzyme that accumulates in many cells undergoing apoptotic cell death. Our data suggest that ligand activation of RARs in HL-60 cells results in a global suppression of BCL-2 expression whereas ligand activation of both RARs and RXRs triggers the selective accumulation of tissue transglutaminase in the apoptotic HL-60 cells.

Activation of retinoid X receptors induces apoptosis in HL-60 cell lines.

Retinoids induce myeloblastic leukemia (HL-60) cells to differentiate into granulocytes, which subsequently die by apoptosis. Retinoid action is mediated through at least two classes of nuclear receptors: retinoic acid receptors, which bind both all-trans retinoic acid and 9-cis retinoic acid, and retinoid X receptors, which bind only 9-cis retinoic acid. Using receptor-selective synthetic retinoids and HL-60 cell sublines with different retinoid responsiveness, we have investigated the contribution that each class of receptors makes to the processes of cellular differentiation and death. Our results demonstrate that ligand activation of retinoic acid receptors is sufficient to induce differentiation, whereas ligand activation of retinoid X receptors is essential for the induction of apoptosis in HL-60 cell lines.

The caliber persistent artery of the stomach: a unifying approach to gastric aneurysm, Dieulafoy's lesion, and submucosal arterial malformation.

The caliber persistent artery of the stomach is the most dangerous form of gastrorrhagias; the overall lethality rate is 60.5%. A literature review aimed at completeness, a study of the hitherto largest case material (24 cases), and a comparative analysis of the bleeding and normal gastric arteries gave the following results: (1) the walls of the pathologic arteries are of normal structure; (2) as submucous arteries, they are of normal diameter; (3) they are attached to the mucosa by virtue of Wanke's musculoelastic mantle; (4) at the level of the muscularis mucosae, they are definitely oversized; (5) in the area of the linkage of the artery to the mucosa, a vulnerable mucosal spot is created; (6) the artery is accompanied by a vein of similar caliber; and (7) perforation of the vein takes place before that of the artery. In addition to surgical pathology and pathogenesis, the pitfalls of its diagnosis and treatment are also discussed. Caliber persistent artery of the stomach is much more common than the literature indicates, necessitating an increased awareness of the condition.

Eosinophilic endomyometritis associated with diagnostic curettage.

Histological study of hysterectomy specimens removed for noninflammatory and nonneoplastic disease revealed 15 severe cases and many more slight cases of eosinophil leukocytic endomyometritis, the degree of which appeared to correlate with injury from preoperative diagnostic curettage. In each case of severe eosinophilic endomyometritis, circumferential mucosal and extensive myometrial damage together with an occluding blood clot at the level of the inner orifice was confirmed. The massive eosinophilia appeared any time between 18 h and 21 days after curettage, and it is proposed that the probable causative agents are eosinophil chemotactic substances liberated from the myometrial mast cells and from the degrading blood clot filling the uterine cavity. There were no clinical signs or symptoms that appeared to correlate with the histological changes observed in this study. This phenomenon may serve as a model for studying the dynamics and functions of eosinophilic leukocytes in humans.