Roles of PPAR transcription factors in the energetic metabolic switch occurring during adult neurogenesis.

PPARs are a class of ligand-activated transcription factors belonging to the superfamily of receptors for steroid and thyroid hormones, retinoids and vitamin D that control the expression of a large number of genes involved in lipid and carbohydrate metabolism and in the regulation of cell proliferation, differentiation and death. The role of PPARs in the CNS has been primarily associated with lipid and glucose metabolism; however, these receptors are also implicated in neural cell differentiation and death, as well as neuronal maturation. Although it has been demonstrated that PPARs play important roles in determining NSCs fate, less is known about their function in regulating NSCs metabolism during differentiation. In order to identify the metabolic events, controlled by PPARs, occurring during neuronal precursor differentiation, the glucose and lipid metabolism was followed in a recognized model of neuronal differentiation in vitro, the SH-SY5Y neuroblastoma cell line. Moreover, PPARs distribution were also followed in situ in adult mouse brains. The concept of adult neurogenesis becomes relevant especially in view of those disorders in which a loss of neurons is described, such as Alzheimer disease, Parkinson disease, brain injuries and other neurological disorders. Elucidating the crucial steps in energetic metabolism and the involvement of PPARγ in NSC neuronal fate (lineage) may be useful for the future design of preventive and/or therapeutic interventions.

Hypoxia induces peroxisome proliferator-activated receptor α (PPARα) and lipid metabolism peroxisomal enzymes in human glioblastoma cells.

Glioblastoma multiforme (GBM) represents the most severe type of glioma, the most common brain tumor. Their malignancy shows a relationship with an increased proliferation and a poorly organized tumor vascularization, an event that leads to inadequate blood supply, hypoxic areas and at last to the formation of necrotic areas, a feature of glioblastoma. Hypoxic/necrotic tumors are more resistant to chemotherapy and radiation therapies, thus it is crucial to formulate new therapeutic approaches that can render these tumors more sensitive to the action of conventional therapies. It has been demonstrated that under hypoxia, gliomas accumulate lipid droplets and that this event is positively correlated with the degree of malignancy, glioblastoma being the most endowed with lipid droplets. We have previously demonstrated in ex vivo glioma specimens a grade-dependent lipid metabolism perturbation. Here we studied the lipid pathways and the presence of stemness markers in glioma primary cultures, obtained from surgical specimens of patients affected by glioma at different grade of malignancy, GBM primary cultures cultured under both hypoxic and normoxic conditions, as well as normal human astrocytes. The results obtained demonstrate that hypoxia plays a crucial role in regulating the expression of lipid metabolism peroxisomal enzymes, the lipid droplets accumulation as well as the transcription factor PPARα.

Signal transduction pathways involved in PPARß/δ-induced neuronal differentiation.

Neuroblastomas are pediatric tumors originating from neuroblasts in the developing peripheral nervous system. The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of survival and differentiation of specific neuronal populations in the central and peripheral nervous system. Patients whose neuroblastoma tumors express high levels of BDNF and TrkB have an unfavorable prognosis. We have previously reported on the neuronal differentiating activity of peroxisome proliferator-activated receptors (PPAR)ß/δ natural and synthetic ligands by modulating BDNF/TrkB pathway, suggesting their potential use as new therapeutic strategies for neuroblastoma. The validation of new therapeutic agents implies the understanding of their mechanisms of action. Herein, we report the effects of activated-PPARß/δ on signal transduction pathways known to be involved in neuronal differentiation, such as ERK1,2 and BDNF pathways. The results obtained, using also PPARß/δ silencing, indicating a neuronal differentiating effect PPARß/δ-dependent through BDNF-P75-ERK1,2 pathways, further support a role for PPARß/δ in neuronal differentiation and pointing towards PPARß/δ as a modulator of pathways crucial for neuronal differentiation. These findings open new perspectives in the formulation of potential therapeutic approaches to be used as adjuvant treatment with the standard therapies.

Lipid metabolism impairment in human gliomas: expression of peroxisomal proteins in human gliomas at different grades of malignancy.

Gliomas are histologically graded by cellularity, cytological atypia, necrosis, mitotic figures, and vascular proliferation, features associated with biologically aggressive behaviour. However, abundant evidence suggests the presence of unrecognized, clinically relevant subclasses of the diffuse gliomas, both in respect to their underlying molecular phenotype and their clinical response to therapy. It is well-known that patient prognosis and therapeutic decisions rely on accurate pathological grading. Recently, it was reported that human gliomas accumulate lipid droplets during progression, suggesting a lipid metabolism impairment. Considering the crucial role of peroxisomes in lipid metabolism, in the present work we studied the expression profiles of proteins either exclusively localized to peroxisomes, such as peroxin14 (PEX14), peroxisomal membrane protein 70Kda (PMP70), acyl-CoA oxidase, thiolase, or partially associated to peroxisomes such as Hydroxymethylglutaryl-CoA reductase (HMGCoA-red) and peroxisomal-related proteins, namely PPARalpha, in human glioma specimens at different grades of malignancy. Moreover, Nile red staining of lipid droplets, thin layer chromatography (TLC) and proton nuclear magnetic resonance spectroscopy (NMR) were carried out in order to correlate the biochemical results with the lipid content of tumor tissues. The results obtained indicate that correlating the malignancy grade with the expression of peroxisomal genes and proteins, may constitute a sensitive tool to highlight possible subtypes not recognized by the classical histological techniques.

Neuronal response of peroxisomal and peroxisome-related proteins to chronic and acute Abeta injury.

The central role of peroxisomes in ROS and lipid metabolism and their importance in brain functioning are well established. The aim of this work was to study the modulation of peroxisomal and peroxisome-related proteins in cortical neurons in vitro challenged with chronic or acute Abeta treatment, in order to investigate whether peroxisomes represent one of the cellular target of Abeta in these cells. The expression of peroxisomal (PMP70, catalase, acyl-CoA oxidase and thiolase), peroxisome-related (PPARalpha, insulin-degrading enzyme) and anti-oxidant (SOD1, SOD2, GSTP1) proteins was studied. The results obtained, demonstrating an early upregulation of the peroxisomal proteins during the chronic challenge, followed by their dramatic impairment after acute challenge, suggest that peroxisomes represent one of the first line of defence against Abeta-mediated oxidative injury. Our results support the notion that substances able to activate PPARalpha and/or to induce peroxisomal proliferation may constitute a novel preventive and/or therapeutic tool against neurodegenerative diseases.

PPARs Expression in Adult Mouse Neural Stem Cells: Modulation of PPARs during Astroglial Differentiaton of NSC.

PPAR isotypes are involved in the regulation of cell proliferation, death, and differentiation, with different roles and mechanisms depending on the specific isotype and ligand and on the differentiated, undifferentiated, or transformed status of the cell. Differentiation stimuli are integrated by key transcription factors which regulate specific sets of specialized genes to allow proliferative cells to exit the cell cycle and acquire specialized functions. The main differentiation programs known to be controlled by PPARs both during development and in the adult are placental differentiation, adipogenesis, osteoblast differentiation, skin differentiation, and gut differentiation. PPARs may also be involved in the differentiation of macrophages, brain, and breast. However, their functions in this cell type and organs still awaits further elucidation. PPARs may be involved in cell proliferation and differentiation processes of neural stem cells (NSC). To this aim, in this work the expression of the three PPAR isotypes and RXRs in NSC has been investigated.

PPARbeta agonists trigger neuronal differentiation in the human neuroblastoma cell line SH-SY5Y.

Neuroblastomas are pediatric tumors originating from immature neuroblasts in the developing peripheral nervous system. Differentiation therapies could help lowering the high mortality due to rapid tumor progression to advanced stages. Oleic acid has been demonstrated to promote neuronal differentiation in neuronal cultures. Herein we report on the effects of oleic acid and of a specific synthetic PPARbeta agonist on cell growth, expression of differentiation markers and on parameters responsible for the malignancy such as adhesion, migration, invasiveness, BDNF, and TrkB expression of SH-SY5Y neuroblastoma cells. The results obtained demonstrate that many, but not all, oleic acid effects are mediated by PPARbeta and support a role for PPARbeta in neuronal differentiation strongly pointing towards PPAR ligands as new therapeutic strategies against progression and recurrences of neuroblastoma.

Immunolocalization of peroxisome proliferator-activated receptors and retinoid X receptors in the adult rat CNS.

Peroxisome proliferator-activated and retinoid X receptors (PPARs and RXRs) are transcription factors belonging to the steroid hormone receptor superfamily. Upon activation by their ligands, PPARs and RXRs bind to their target genes as heterodimers. Ligands of these receptors include lipophylic molecules, such as retinoids, fatty acids and eicosanoids, the importance of which in the metabolism and functioning of the nervous tissue is well documented. The immunohistochemical distribution of PPARs and RXRs in the CNS of the adult rat was studied by means of a sensitive biotinyl-tyramide method. All PPAR (alpha, beta/delta and gamma) and RXR (alpha, beta and gamma) isotypes were detected and found to exhibit specific patterns of localization in the different areas of the brain and spinal cord. The presence of the nuclear receptors was observed in both neuronal and glial cells. While PPAR beta/delta and RXR beta showed a widespread distribution, alpha and gamma isotypes exhibited a more restricted pattern of expression. The frontal cortex, basal ganglia, reticular formation, some cranial nerve nuclei, deep cerebellar nuclei, and cerebellar Golgi cells appeared rather rich in all studied receptors. Based on our data, we suggest that in the adult CNS, PPARs and RXRs, besides playing roles common to many other tissues, may have specific functions in regulating the expression of genes involved in neurotransmission, and therefore play roles in complex processes, such as aging, neurodegeneration, learning and memory.

Influence of a low background radiation environment on biochemical and biological responses in V79 cells.

We present the results of an experiment aimed at comparing the effects of different background radiation environments on metabolism and responses to gamma-rays and cycloheximide of cultured mammalian cells. Chinese hamster V79 cells were maintained in exponential growth in parallel for up to 9 months at the Istituto Superiore di Sanità (ISS) and at the INFN-Gran Sasso underground Laboratory (LNGS) where exposure due to gamma-rays and to radon was reduced by factors of about 70 and 25, respectively. After 9 months the cells grown at the LNGS (cumulative gamma dose about 30 microGy, average radon concentration around 5 Bq/m(3)), compared to the cells grown at the ISS (cumulative gamma-ray dose about 2 mGy, average radon concentration around 120 Bq/m(3)), exhibited i). a significant increase of the cell density at confluence, ii). a significantly higher capacity to scavenge organic and inorganic hydroperoxides but a reduced scavenging capacity towards superoxide anions and iii). an increase in both the basal hprt mutation frequency and sensitivity to the mutagenic effect of gamma-rays. The cells grown at the LNGS also showed a greater apoptotic sensitivity starting at the third month of culture, that was no longer detected after 9 months. Overall, these data suggest a role of background ionizing radiation in determining an adaptive response, although they cannot be considered conclusive.

Immunocytochemical localization of acyl-CoA oxidase in the rat central nervous system.

Peroxisomal beta-oxidation, consisting of four steps catalysed by an acyl-CoA oxidase, a multifunctional protein and a thiolase, is responsible for the shortening of a variety of lipid compounds. The first reaction of this pathway is catalysed by a FAD-containing acyl-CoA oxidase, three isotypes of which have been so far recognised. Among these, straight-chain acyl-CoA oxidase (ACOX) acts on long and very long chain fatty acids, prostaglandins and some xenobiotics. We investigated ACOX localisation by means of a sensitive, tyramide based, immunocytochemical technique, thus obtaining a complete distribution atlas of the enzyme in adult rat CNS. Granular immunoreaction product was found in the cytoplasm of neuronal and glial cells, both in the perikarya and in the cell processes. ACOX immunoreactive neurons were present to variable extent, in either forebrain or hindbrain areas. Specifically, the strongest signal was detected in the pallidum, septum, red nucleus, reticular formation, nuclei of the cranial nerves, and motoneurons of the spinal cord. We then compared the ACOX immunoreactivity pattern with our previous distribution maps of other peroxisomal enzymes in the adult rat brain. While ACOX appeared to colocalise with catalase in the majority of cerebral regions, some differences with respect to d-amino acid oxidase were noted. These observations support the hypothesis of heterogeneous peroxisomal populations in the nervous tissue. The wide distribution of the enzyme in the brain is consistent with the severe and generalised neurological alterations characterising the peroxisomal disorder caused by ACOX deficiency (pseudo-neonatal adrenoleukodystrophy).

Peroxisome proliferator-activated receptors (PPARs) and peroxisomes in rat cortical and cerebellar astrocytes.

Astrocytes are the most versatile cells of the neural tissue. Numerous astrocytic functions--such as protection from oxidative damage, catabolism of neuroactive D-amino acids acting as neuromodulators, synthesis and catabolism of some lipid molecules, and, possibly, gluconeogenesis--reside in peroxisomes. The expression of several peroxisomal enzymes, particularly those of the acyl-CoA beta-oxidation pathway, is regulated by a class of ligand-activated transcription factors, known as peroxisome proliferator-activated receptors (PPARs), acting on their target genes as heterodimers with the retinoid X receptors (RXRs). In this work, primary and secondary cultures of astrocytes from the cerebral cortices and cerebella of neonatal rats (2 and 7 days of postnatal age) were utilized to investigate the expression of peroxisomal enzymes, PPAR and RXR isotypes (alpha, beta and gamma), by both biochemical and immunological methods. The results obtained demonstrate that astrocytes in vitro express peroxisomal enzymes, PPARs, and RXRs and that differences dependent on brain area, animal age, and culture time are reminiscent of the in vivo situation. Therefore, primary cultures of astrocytes and, particularly, high purified subcultures may constitute a useful model for further studies aimed to gain further insights into the roles of peroxisomes and PPARs related to lipid and glucose metabolism in these cells.

Presence and inducibility of peroxisomes in a human glioblastoma cell line.

We investigated the effect of the peroxisomal proliferator (PP) perfluorodecanoic acid (PFDA), alone or in combination with 9-cis-retinoic acid (RX) on the human glioblastoma cell line Lipari (LI). Cell proliferation, apoptotic rate, peroxisome morphology and morphometry, peroxisomal enzyme activities and the presence of peroxisome proliferator-activated receptors (PPARs) were examined. We show that PFDA alone produces pleiotropic effects on LI cells and that RX enhances some of these effects. Peroxisomal number and relative volume, as well as palmitoyl-CoA oxidase activity and protein, are increased by PFDA treatment, with a synergistic effect by RX. The latter, alone or in association with PFDA, induces catalase activity and protein, increases apoptosis and decreases cell proliferation. PPAR isotypes alpha and gamma were detected in LI cells. While the former is apparently unaffected by either treatment, the latter increases in response to PFDA, independent of the presence of RX. The results of this study are discussed in terms of PPARalpha activation and PPARgamma induction by PFDA, by either a direct or an indirect mechanism.

Immunocytochemical localization of D-amino acid oxidase in rat brain.

D-amino acid oxidase (D-AAO) is a peroxisomal flavoenzyme, the physiological substrate and the precise function of which are still unclear. We have investigated D-AAO distribution in rat brain, by immunocytochemistry, with an affinity-purified polyclonal antibody. Immunoreactivity occurred in both neuronal and glial cells, albeit at different densities. Glial immunostaning was strongest in the caudal brainstem and cerebellar cortex, particularly in astrocytes, Golgi-Bergmann glia, and tanycytes. Hindbrain neurons were generally more immunoreactive than those in the forebrain. Immunopositive forebrain cell populations included mitral cells in the olfactory bulb, cortical and hippocampal neurons, ventral pallidum, and septal, reticular thalamic, and paraventricular hypothalamic nuclei. Within the positive regions, not all the neuronal populations were equally immunoreactive; for example, in the thalamus, only the reticular and anterodorsal nuclei showed intense labelling. In the hindbrain, immunopositivity was virtually ubiquitous, and was especially strong in the reticular formation, pontine, ventral and dorsal cochlear, vestibular, cranial motor nuclei, deep cerebellar nuclei, and the cerebellar cortex, especially in Golgi and Purkinje cells.

Presence of heterogeneous peroxisomal populations in the rat nervous tissue.

Peroxisomes were purified from the nervous tissue of 14-day-old rats by means of a Nycodenz gradient. Peroxisomal enzymes exhibited different sedimentation patterns: dihydroxyacetone phosphate acyl-transferase equilibrates at 1.142 g/ml together with the first peak of catalase; palmitoyl-CoA oxidase and D-amino acid oxidase activities are mainly recovered at 1.154 g/ml; the second peak of catalase is found at 1.175 g/ml. Morphological and semi-quantitative analyses of immunogold-labelled peroxisomes reveal profound heterogeneity of the particles. Very small (=0.2 microm diameter), electron dense vesicles containing catalase or thiolase, but devoid of other tested enzymes, are preferentially found in the light region, together with larger ( > 0.2 < 0.3 microm) and less electron dense palmitoyl-CoA oxidase-positive peroxisomes. At intermediate density (1.154 g/ml) peroxisomes of more uniform size (0.25-0.27 microm), containing palmitoyl-CoA oxidase or thiolase with or without catalase are preferentially found. This population extends toward the densest region of the gradient, where very large D-amino acid oxidase-containing peroxisomes are also found. In this region, smaller peroxisomes, often polymorphic, which are catalase- and thiolase-positive and D-amino acid oxidase/palmitoyl-CoA oxidase-negative, are also observed. The possibility that the heterogeneity of neural peroxisomes may reflect both cellular heterogeneity and ongoing peroxisomal biogenesis is discussed.

Lysosomal involvement in the removal of clofibrate-induced rat liver peroxisomes. A biochemical and morphological analysis.

Peroxisomal proliferators induce in rodents hepatic hyperplasia and hypertrophy; the significant increase in the peroxisomal population is accompanied by specific and reversible induction of some peroxisomal enzymes. In suckling rats born from clofibrate-treated mothers, a massive removal of proliferated organelles occurs within 3 days of recovery. In the present paper we examined the early stages of the recovery period in liver of male rats treated with clofibrate for 5 days. The lysosomal involvement in the removal of drug-induced peroxisomes was investigated under physiological conditions, i.e. in the absence of inhibitors of the autophagic process. Biochemical results indicate that peroxisomal beta-oxidation, but not catalase activity, returns to the control values within the examined period. Total acid phosphatase activity is not affected by clofibrate treatment, but following fractionation on a linear density gradient the lysosomal marker enzyme activity is shifted towards lower density values, particularly at day 1 and 2 of recovery. This class of organelles possibly represents lysosomes involved in active autophagic processes. Acid phosphatase cytochemistry shows an increase of lysosome number at day 1 of recovery. Combination of acid phosphatase cytochemistry either with catalase cytochemistry or with catalase immunogold labelling allows to reveal organelles containing both marker enzymes. These results strongly support the involvement of autophagic processes in the removal of proliferated peroxisomes.

Regional and ultrastructural immunolocalization of copper-zinc superoxide dismutase in rat central nervous system.

We examined the distribution of copper-zinc superoxide dismutase (CuZnSOD) in adult rat central nervous system by light and electron microscopic immunocytochemistry, using an affinity-purified polyclonal antibody. The enzyme appeared to be exclusively localized in neurons. No immunoreactivity was seen in non-neuronal cells. The staining intensity was variable, depending on the brain region and, within the same region, on the neuron type. Highly immunoreactive elements included cortical neurons evenly distributed in the different layers, hippocampal interneurons, neurons of the reticular thalamic nucleus, and Golgi, stellate, and basket cells of the cerebellar cortex. Other neurons, i.e., pyramidal cells of the neocortex and hippocampus, Purkinje and granule cells of the cerebellar cortex, and the majority of thalamic neurons, showed much weaker staining. In the spinal cord, intense CuZnSOD immunoreactivity was present in many neurons, including motor neurons. Pre-embedding immunoelectron microscopy of the neocortex, hippocampus, reticular thalamic nucleus, and cerebellar cortex showed cytosolic and nucleoplasmic labeling. Moreover, single membrane-limited immunoreactive organelles identified as peroxisomes were often found, even in neurons that appeared weakly stained at the light microscopic level. In double immunogold labeling experiments, particulate CuZnSOD immunoreactivity co-localized with catalase, a marker enzyme for peroxisomes, thus demonstrating that in neural tissue CuZnSOD is also present in peroxisomes.

Immunocytochemical localization of catalase in the central nervous system of the rat.

Catalase is a marker for peroxisomes, which are ubiquitous cytoplasmic organelles. Although the distribution and features of peroxisomes are well known in liver and kidney, these organelles have been rarely studied in neural tissues. Catalase is an important scavenging enzyme against reactive oxygen species, as it removes H2O2 produced during metabolic processes. Reactive oxygen species are involved in a number of brain lesions and in brain aging. We investigated the distribution of catalase in rat central nervous system by means of a newly developed immunocytochemical procedure for signal enhancement, using an affinity-purified polyclonal antiserum. The data show that catalase immunoreactivity is present in all neural cells, both neuronal and glial, albeit at different concentrations. Among glial cells, ependymal cells and tanycytes of the third ventricle and the median eminence show the most intense immunoreaction; positivity is also found in oligodendrocytes and astrocytes. In general, neurons in the brainstem are relatively more immunoreactive than those in the forebrain although, within these respective brain regions, there are areas with low and high staining intensity. Moreover, within the same area, certain types of neuron appear more immunoreactive than others. The cell bodies in the septal nuclei, pallidum, reticular thalamic nucleus, mesencephalic nucleus of the trigeminal nerve, Deiter's nucleus, locus ceruleus, cranial and spinal motor nuclei, and the Golgi cells of the cerebellar cortex are among the most densely stained neurons. Catalase immunoreactivity of the cell bodies, which presumably is proportional to catalase content, appears to be only partially correlated with cell size or type of neurotransmitter used in the nerve endings; it is likely that other unknown parameters regulate the abundance of the enzyme. In many cases, highly immunoreactive cells correspond to neurons known to be resistant to ischemia-reperfusion injury, whereas weakly stained cells correspond to neurons that are more susceptible to ischemic damage. The amount of catalase may be critical for a protective effect against oxidative stress under pathological conditions, such as ischemia-reperfusion injury.

Effects of Di-(2-ethylhexyl)phthalate on peroxisomes of liver, kidney and brain of lactating rats and their pups.

Di-(2-ethylhexyl)phthalate administered to adult lactating rats, from delivery to weaning, induces modifications of the peroxisomal enzymatic pattern in the liver, kidney and brain of both dams and pups. These modifications are age- and organ-dependent. Biochemical analysis shows that: 1) catalase specific activity is two-fold increased in the liver of both adult and newborn animals, in the kidney of newborns and in the brain of adults. 2)D-amino acid oxidase doubles in all newborn organs and in adult brain; it increases, although to a lesser extent, also in adult kidney, while it is half-reduced in adult liver. 3) Dihydroxyacetone phosphate acyl transferase only doubles in newborn liver, remaining fairly unchanged in all the other tested tissues. 4) Palmitoyl-CoA oxidase is greatly induced in the liver of both dams and litters, doubled in the kidneys and slightly increased or not at all in the brain of pups and mothers, respectively. The effect of the drug on enzyme activities is reversible, with different time courses depending on the considered enzyme and organ. Western blottings confirm the biochemical data. Electron microscopy shows proliferated peroxisomes in the liver and kidney of treated animals but not in the brain, where high catalase-like immunoreactivity is observed in the cytosol of neurons. Taken together, our data demonstrate that the response of peroxisomal enzymes to DEHP treatment is age- as well as tissue-dependent and specific for each enzyme studied.

Purification of peroxisomal fraction from rat brain.

A purification procedure to obtain peroxisomes (microperoxisomes) from the brain of suckling rats is reported. A P2 fraction, (crude light mitochondria) frozen and thawed seven times, was subfractionated yielding a P4 fraction, 4-fold enriched in catalase activity with respect to the cytoplasmic extract S1. The P4 fraction was used for further purification of peroxisomes by isopicnic centrifugation on Nycodenz gradient (1.10-1.20 g/ml). When the cerebellum was not included in the starting material, the equilibrium density of peroxisomes was 1.152-1.162 g/ml. In this case the overall yield of catalase in the most enriched fraction was 7% and its relative specific activity more than 50. When the cerebellum was included in the total homogenate, the equilibrium density shifted towards higher values (1.177 g/ml) and in this case the catalase relative specific activity in the peroxisomal enriched fraction was extremely high (> 100). The biochemical results, together with the electron microscope examination of the purified fractions, demonstrate that our procedure allows the best purification of brain peroxisomes so far obtained. The different equilibrium densities of peroxisomes observed in the two sets of experiments are interpreted in terms of size heterogeneity of these organelles in different brain portions and cell types.

In vivo and in vitro induction of 'tissue' transglutaminase in rat hepatocytes by retinoic acid.

Tissue transglutaminase (tTG) expression was found to be induced in rat liver following in vivo retinoic acid (RA) treatment (Piacentini et al. (1988) Biochem. J. 253, 33-38). Here we show that the increased enzyme expression in rat liver is at least partially the result of the action of RA in parenchymal cells. In fact, (a) when hepatocytes are isolated from RA-treated animals their transglutaminase protein content is much higher than in similarly isolated control cells; (b) higher tTG protein level is also found by immunoelectronmicroscopy in the hepatocytes of the RA-treated rats as compared with the very low amount detected in the controls; (c) RA induces tTG in hepatocytes under culture conditions as well. One of the functions of tTG is to form a protein polymer in dying apoptotic cells by epsilon(gamma-glutamyl)lysine and, specifically gamma-glutamylpolyamine cross-links (Fesus et al. (1989) FEBS Lett. 245, 150-154). Noteworthy, after in vivo and in vitro RA-treatment we could not determine any increase (there was even a slight decrease) in the number of the cross-linked apoptotic envelopes. In keeping with this is the significant reduction of protein bound gamma-glutamylpolyamine detected in hepatocytes exposed to RA in culture. These findings suggest that the RA-induced tTG in parenchimal cells is an inactive form.