RESUMO
Cancer progression is associated with epigenetic alterations, such as changes in DNA methylation, histone modifications or variants incorporation. The p400 ATPase, which can incorporate the H2A.Z variant, and the Tip60 histone acetyltransferase are interacting chromatin-modifying proteins crucial for the control of cell proliferation. We demonstrate here that Tip60 acts as a tumor suppressor in colon, since mice heterozygous for Tip60 are more susceptible to chemically induced preneoplastic lesions and adenomas. Strikingly, heterozygosity for p400 reverses the Tip60-dependent formation of preneoplastic lesions, uncovering for the first time pro-oncogenic functions for p400. By genome-wide analysis and using a specific inhibitor in vivo, we demonstrated that these effects are dependent on Wnt signaling which is antagonistically impacted by p400 and Tip60: p400 directly favors the expression of a subset of Wnt-target genes and regulators, whereas Tip60 prevents ß-catenin acetylation and activation. Taken together, our data underline the physiopathological importance of interplays between chromatin-modifying enzymes in the control of cancer-related signaling pathways.
Assuntos
Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Histona Acetiltransferases/fisiologia , Histonas/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Transativadores/fisiologia , Proteínas Wnt/metabolismo , Acetilação , Animais , Western Blotting , Células Cultivadas , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Neoplasias do Colo/genética , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Lisina Acetiltransferase 5 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMO
In proliferating myoblasts, muscle specific genes are silenced by epigenetic modifications at their promoters, including histone H3K9 methylation. Derepression of the promoter of the gene encoding the myogenic factor myogenin (Myog) is key for initiation of muscle differentiation. The mechanism of H3K9 demethylation at the Myog promoter is unclear, however. Here, we identify an isoform of the histone demethylase JMJD2A/KDM4A that lacks the N-terminal demethylase domain (ΔN-JMJD2A). The amount of ΔN-JMJD2A increases during differentiation of C2C12 myoblasts into myotubes. Genome-wide expression profiling and exon-specific siRNA knockdown indicate that, in contrast to the full-length protein, ΔN-JMJD2A is necessary for myotube formation and muscle-specific gene expression. Moreover, ΔN-JMJD2A promotes MyoD-induced conversion of NIH3T3 cells into muscle cells. ChIP-on-chip analysis indicates that ΔN-JMJD2A binds to genes mainly involved in transcriptional control and that this binding is linked to gene activation. ΔN-JMJD2A is recruited to the Myog promoter at the onset of differentiation. This binding is essential to promote the demethylation of H3K9me2 and H3K9me3. We conclude that induction of the ΔN-JMJD2A isoform is crucial for muscle differentiation: by directing the removal of repressive chromatin marks at the Myog promoter, it promotes transcriptional activation of the Myog gene and thus contributes to initiation of muscle-specific gene expression.
Assuntos
Diferenciação Celular , Histona Desmetilases/fisiologia , Músculo Esquelético/citologia , Animais , Células Cultivadas , Cromatina/metabolismo , Epigênese Genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Camundongos , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Células NIH 3T3 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , TransfecçãoRESUMO
The histone variant H2A.Z plays important functions in the regulation of gene expression. In mammals, it is encoded by two genes, giving rise to two highly related isoforms named H2A.Z.1 and H2A.Z.2, which can have similar or antagonistic functions depending on the promoter. Knowledge of the physiopathological consequences of such functions emerges, but how the balance between these isoforms regulates tissue homeostasis is not fully understood. Here, we investigated the relative role of H2A.Z isoforms in intestinal epithelial homeostasis. Through genome-wide analysis of H2A.Z genomic localization in differentiating Caco-2 cells, we uncovered an enrichment of H2A.Z isoforms on the bodies of genes which are induced during enterocyte differentiation, stressing the potential importance of H2A.Z isoforms dynamics in this process. Through a combination of in vitro and in vivo experiments, we further demonstrated the two isoforms cooperate for stem and progenitor cells proliferation, as well as for secretory lineage differentiation. However, we found that they antagonistically regulate enterocyte differentiation, with H2A.Z.1 preventing terminal differentiation and H2A.Z.2 favoring it. Altogether, these data indicate that H2A.Z isoforms are critical regulators of intestine homeostasis and may provide a paradigm of how the balance between two isoforms of the same chromatin structural protein can control physiopathological processes.
RESUMO
The p400 E1A-associated protein, which mediates H2A.Z incorporation at specific promoters, plays a major role in cell fate decisions: it promotes cell cycle progression and inhibits induction of apoptosis or senescence. Here, we show that p400 expression is required for the correct control of ROS metabolism. Depletion of p400 indeed increases intracellular ROS levels and causes the appearance of DNA damage, indicating that p400 maintains oxidative stress below a threshold at which DNA damages occur. Suppression of the DNA damage response using a siRNA against ATM inhibits the effects of p400 on cell cycle progression, apoptosis, or senescence, demonstrating the importance of ATM-dependent DDR pathways in cell fates control by p400. Finally, we show that these effects of p400 are dependent on direct transcriptional regulation of specific promoters and may also involve a positive feedback loop between oxidative stress and DNA breaks since we found that persistent DNA breaks are sufficient to increase ROS levels. Altogether, our results uncover an unexpected link between p400 and ROS metabolism and allow deciphering the molecular mechanisms largely responsible for cell proliferation control by p400.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Homeostase , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Dano ao DNA , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , RNA Interferente Pequeno/genética , Transdução de Sinais , Transcrição GênicaRESUMO
In the intestinal epithelium, the Cdx, GATA, and HNF transcription factor families are responsible for the expression of differentiation markers such as sucrase-isomaltase. Although previous studies have shown that Cdx2 can induce differentiation in rat intestinal IEC-6 cells, no data are available concerning the direct implication of transcription factors on differentiation in human normal intestinal epithelial cell types. We investigated the role of Cdx2, GATA-4, and HNF-1alpha using the undifferentiated human intestinal epithelial crypt cell line HIEC. These transcription factors were tested on proliferation and expression of polarization and differentiation markers. Ectopic expression of Cdx2 or HNF-1alpha, alone or in combination, altered cell proliferation abilities through the regulation of cyclin D1 and p27 expression. HNF-1alpha and GATA-4 together induced morphological modifications of the cells toward polarization, resulting in the appearance of functional features such as microvilli. HNF-1alpha was also sufficient to induce the expression of cadherins and dipeptidylpeptidase, whereas in combination with Cdx2 it allowed the expression of the late differentiation marker sucrase-isomaltase. Large-scale analysis of gene expression confirmed the cooperative effect of these factors. Finally, although DcamKL1 and Musashi-1 expression were downregulated in differentiated HIEC, other intestinal stem cell markers, such as Bmi1, were unaffected. These observations show that, in cooperation with Cdx2, HNF-1alpha acts as a key factor on human intestinal cells to trigger the onset of their functional differentiation program whereas GATA-4 appears to promote morphological changes.
Assuntos
Diferenciação Celular/fisiologia , Enterócitos/citologia , Fator de Transcrição GATA4/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/citologia , Células-Tronco/citologia , Fator de Transcrição CDX2 , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Proliferação de Células , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p27 , Dipeptidil Peptidase 4/genética , Quinases Semelhantes a Duplacortina , Regulação para Baixo/genética , Enterócitos/metabolismo , Enterócitos/ultraestrutura , Fator de Transcrição GATA4/genética , Expressão Gênica/genética , Perfilação da Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/genética , Humanos , Interfase/genética , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1 , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a RNA/genética , Receptores Acoplados a Proteínas G/genética , Proteínas Repressoras/genética , Células-Tronco/metabolismo , Células-Tronco/ultraestrutura , Complexo Sacarase-Isomaltase/genética , Transfecção , Regulação para Cima/genéticaRESUMO
The apoptotic process is accompanied by major changes in chromatin structure and gene expression. The apoptotic genetic program is progressively set up with the inhibition of antiapoptotic genes and the activation of proapoptotic ones. Here, we show that the histone deacetylase 3 (HDAC-3), which is a known co-repressor of many proapoptotic genes, is subjected to proteolytic cleavage during apoptosis in a cell type- and species-independent manner. This cleavage is caspase dependent and leads to the loss of the C-terminal part of HDAC-3. The cleaved form of HDAC-3 accumulates in the cytoplasm. Furthermore, we found that forced nuclear localization of HDAC-3 decreases the efficiency of apoptosis induction, indicating that HDAC-3 cytoplasmic relocalization is important for the apoptotic process. Finally, we observed that HDAC-3 cleavage allowed increased histone acetylation and transcriptional activation on a proapoptotic HDAC-3-target gene, the Fas-encoding gene. Altogether, our results thus indicate that HDAC-3 cleavage is crucial for efficient apoptosis induction because it allows the activation of some proapoptotic genes during apoptosis progression.
Assuntos
Apoptose , Histona Desacetilases/metabolismo , Acetilação , Transporte Ativo do Núcleo Celular , Caspases/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Histonas/metabolismo , Humanos , Células Jurkat , Transporte Proteico , Ativação Transcricional , Receptor fas/metabolismoRESUMO
The rapid renewal of intestinal epithelium is mediated by a pool of stem cells, located at the bottom of crypts, giving rise to highly proliferative progenitor cells, which in turn differentiate during their migration along the villus. The equilibrium between renewal and differentiation is critical for establishment and maintenance of tissue homeostasis, and is regulated by signaling pathways (Wnt, Notch, Bmp ) and specific transcription factors (TCF4, CDX2 ). Such regulation controls intestinal cell identities by modulating the cellular transcriptome. Recently, chromatin modification and dynamics have been identified as major actors linking signaling pathways and transcriptional regulation in the control of intestinal homeostasis. In this review, we synthesize the many facets of chromatin dynamics involved in controlling intestinal cell fate, such as stemness maintenance, progenitor identity, lineage choice and commitment, and terminal differentiation. In addition, we present recent data underlying the fundamental role of chromatin dynamics in intestinal cell plasticity. Indeed, this plasticity, which includes dedifferentiation processes or the response to environmental cues (like microbiota's presence or food ingestion), is central for the organ's physiology. Finally, we discuss the role of chromatin dynamics in the appearance and treatment of diseases caused by deficiencies in the aforementioned mechanisms, such as gastrointestinal cancer, inflammatory bowel disease or irritable bowel syndrome. Graphical abstract.
Assuntos
Linhagem da Célula , Plasticidade Celular , Cromatina/metabolismo , Homeostase , Mucosa Intestinal/metabolismo , Animais , Histonas/metabolismo , HumanosRESUMO
Permanent silencing of E2F-dependent genes is a hallmark of the irreversible cell cycle exit that characterizes terminally differentiated and senescent cells. The determinant of this silencing during senescence has been proposed to be the binding of the retinoblastoma protein Rb and the consequent methylation of H3K9. During ex vivo skeletal muscle differentiation, while most cells terminally differentiate and form myotubes, a subset of myoblasts remains quiescent and can be reinduced by growth factor stimulation to enter the cell cycle. Thus, differentiating cells are composed of two different populations: one in which E2F-dependent genes are permanently repressed and the other not. We observed that, in a manner reminiscent to senescent cells, permanent silencing of the E2F-dependent cdc6, dhfr, and p107 promoters in myotubes was associated with a specific increase in H3K9 trimethylation. To investigate the role of Rb in this process, we developed a reliable method to detect Rb recruitment by chromatin immunoprecipitation. Surprisingly, we observed that Rb was recruited to these promoters more efficiently in quiescent cells than in myotubes. Thus, our data indicate that during muscle differentiation, permanent silencing and H3K9 trimethylation of some E2F-dependent genes are not directly specified by Rb binding, in contrast to what is proposed for senescence.
Assuntos
Fatores de Transcrição E2F/metabolismo , Músculo Esquelético/fisiologia , Mioblastos/fisiologia , Regiões Promotoras Genéticas , Proteína do Retinoblastoma/metabolismo , Animais , Ciclo Celular , Diferenciação Celular , Linhagem Celular , Senescência Celular , Cromatina/metabolismo , Amplificação de Genes , Camundongos , Músculo Esquelético/citologia , Mioblastos/citologia , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The integrin alpha6 subunit exists as two different variants, termed alpha6A and alpha6B. These two variants have been shown to harbor potentially distinct biochemical properties but little is known about their cellular function. The aim of this work was to characterize the expression of the integrin alpha6A and B variants in relation to cell proliferation and differentiation in the human small intestinal epithelium. The results showed distinct expression patterns for the two variants along the crypt-villus axis. Indeed, proliferative cells of the crypt were found to predominantly express alpha6A, while differentiated enterocytes and Paneth cells expressed the alpha6B variant. A similar relationship was observed in intestinal cell models by competitive RT-PCR. Further studies in the Caco-2 cell model showed that manipulating the cellular balance of the two alpha6 variants can influence transcriptional activities related to cell proliferation but not differentiation. This suggests that differential expression of the alpha6 subunits is involved in the intestinal epithelial cell renewal process. Further studies will be needed to substantiate this hypothesis.
Assuntos
Diferenciação Celular/fisiologia , Integrina alfa6/metabolismo , Integrina beta4/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Células CACO-2 , Proliferação de Células , Humanos , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Isoformas de Proteínas/metabolismoRESUMO
The Tip60/p400 chromatin-modifying complex, which is involved in the incorporation and post-translational modification of the H2A.Z histone variant, regulates cell proliferation and important signaling pathways, such as Wnt. Here, we study the involvement of H2A.Z in intestinal epithelial homeostasis, which is dependent on the finely-tuned equilibrium between stem cells renewal and differentiation, under the control of such pathway. We use cell models and inducible knock-out mice to study the impact of H2A.Z depletion on intestinal homeostasis. We show that H2A.Z is essential for the proliferation of human cancer and normal intestinal crypt cells and negatively controls the expression of a subset of differentiation markers, in cultured cells and mice. H2A.Z impairs the recruitment of the intestine-specific transcription factor CDX2 to chromatin, is itself a target of the Wnt pathway and thus, acts as an integrator for Wnt signaling in the control of intestinal epithelial cell fate and homeostasis.
Assuntos
Histonas/metabolismo , Homeostase/genética , Mucosa Intestinal/fisiologia , Via de Sinalização Wnt/genética , Animais , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Células CACO-2 , Diferenciação Celular/genética , Proliferação de Células/genética , Cromatina/genética , Células Epiteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Células HCT116 , Histonas/genética , Humanos , Mucosa Intestinal/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Since chromatin-modifying enzymes are involved in most processes needing to access the DNA fiber such as transcription, replication or DNA repair, their involvement in the regulation of gene expression in numerous physiopathological contexts is widely studied. Most of these enzymes are essential for cell growth and survival due to their pleiotropic roles and studying their impact in vivo on organ development or tissue physiopathology is challenging. In this chapter, we describe a chemically-mediated method to induce colorectal carcinogenesis that we have used to identify in vivo the role of two chromatin modifying enzymes belonging to the same multimolecular complex, the histone acetyltransferase Tip60 and the histone variant-incorporating ATPase p400.
Assuntos
Cromatina/metabolismo , Neoplasias Colorretais/patologia , Lisina Acetiltransferase 5/metabolismo , Neoplasias Experimentais/patologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Azoximetano/toxicidade , Benzenoacetamidas/farmacologia , Carcinogênese/induzido quimicamente , Carcinogênese/patologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/induzido quimicamente , DNA Helicases , Proteínas de Ligação a DNA , Sulfato de Dextrana/toxicidade , Histonas/metabolismo , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lisina Acetiltransferase 5/genética , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/induzido quimicamente , Piridinas/farmacologia , Transativadores/genética , Fatores de Transcrição/genética , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Pericentric heterochromatin is a highly compacted structure required for accurate chromosome segregation in mitosis. In mammals, it relies on methylation of histone H3K9 by Suv39H enzymes, which provides a docking site for HP1 proteins, therefore mediating heterochromatin compaction. Here we show that, when this normal compaction pathway is defective, the histone acetyltransferase Tip60 is recruited to pericentric heterochromatin, where it mediates acetylation of histone H4K12. Furthermore, in such a context, depletion of Tip60 leads to derepression of satellite transcription, decompaction of pericentric heterochromatin, and defects in chromosome segregation in mitosis. Finally, we show that depletion of BRD2, a double bromodomain-containing protein that binds H4K12ac, phenocopies the Tip60 depletion with respect to heterochromatin decompaction and defects in chromosome segregation. Taking the results together, we identify a new compaction pathway of mammalian pericentric heterochromatin relying on Tip60 that might be dependent on BRD2 recruitment by H4K12 acetylation. We propose that the underexpression of Tip60 observed in many human tumors can promote genetic instability via defective pericentric heterochromatin.
Assuntos
Instabilidade Cromossômica/genética , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Acetilação , Animais , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Histona Acetiltransferases/genética , Lisina Acetiltransferase 5 , Metilação , Metiltransferases/genética , Camundongos , Mitose/genética , Células NIH 3T3 , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Transdução de Sinais , Transativadores/genética , Fatores de TranscriçãoRESUMO
The homeobox gene Cdx2 is involved in the regulation of the expression of intestine specific markers such as sucrase-isomaltase and lactase-phlorizin hydrolase. Previous studies performed with immortalized or transformed intestinal cell lines have provided evidence that Cdx2 can promote morphological and functional differentiation in these experimental models. However, no data exist concerning the implication of this factor in normal human intestinal cell physiology. In the present work, we have investigated the role of Cdx2 in normal human intestinal epithelial crypt (HIEC) cells that lack this transcription factor. The establishment of HIEC cells expressing Cdx2 in an inducible manner shows that forced expression of Cdx2 significantly alters the proliferation of intestinal crypt cells and stimulates dipeptidylpeptidase IV expression but is not sufficient to trigger intestinal terminal differentiation. These observations suggest that Cdx2 requires additional factors to activate the enterocyte differentiation program in normal undifferentiated cells.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/citologia , Biomarcadores , Fator de Transcrição CDX2 , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Dipeptidil Peptidase 4/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Oligo-1,6-Glucosidase/metabolismo , Sacarose/metabolismoRESUMO
In epithelia, abnormal expression of E-cadherin is related to pathologies involving a loss of cell polarization and/or differentiation. However, recent observations suggest that E-cadherin could also be repressed under physiological conditions, such as in some epithelial stem cell lineages. In the present work, we have analyzed E-cadherin expression in human intestinal epithelial cell progenitors and investigated its potential role. E-cadherin expression was analyzed along the crypt-villus axis by immunofluorescence on cryosections of small intestine. E-cadherin was found to be differentially expressed, being significantly weaker in the cells located at the bottom of the crypts. Surprisingly, neither the E-cadherin protein nor transcript were detected in a normal human intestinal epithelial (HIEC) crypt cell model isolated in our laboratory, whereas other E-cadherin-related components such as catenins and APC were present. Forced expression of E-cadherin in HIEC cells increased membrane-associated beta-catenin and was accompanied by the appearance of junction-like structures at the cell-cell interface. Functionally, cell kinetics and p21Cip levels were found to be altered in the E-cadherin expressing HIEC cells as compared to controls. Furthermore, a significant reduction of the migration abilities and an increase in sensitivity to anoikis were also observed. These results suggest that down-regulated expression of E-cadherin is a human intestinal crypt base cell-related feature that appears to be of functional relevance for the maintenance of the progenitor cell population.
Assuntos
Biomarcadores/metabolismo , Caderinas/metabolismo , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Adenoviridae/genética , Anoikis , Western Blotting , Células CACO-2 , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Divisão Celular , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica , Genes APC , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestrutura , Intestino Delgado/química , Cinética , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Transativadores/metabolismo , beta CateninaRESUMO
Laminins are basement membrane molecules that mediate cell functions such as adhesion, proliferation, migration, and differentiation. In the normal small intestine, laminin-5 and -10 are mainly expressed at the base of villus cells. However, in Crohn's disease (CD), a major redistribution of these laminins to the crypt region of the inflamed ileal mucosa has been observed, suggesting a possible relationship between laminin expression and cytokine and/or growth factor production, which is also altered in CD. The aim of this study was to test the hypothesis that proinflammatory cytokines can modulate laminin expression by intestinal epithelial cells. The effect of TNF-alpha, IFN-gamma, IL-1beta, IL-6, and transforming growth factor (TGF)-beta was analyzed on the expression of laminins in the normal human intestinal epithelial crypt (HIEC) cell line. When treated with a single cytokine, HIEC cells secreted small amounts of laminin-5 and -10. Only TNF-alpha and TGF-beta induced a slight increase in the secretion of these laminins. However, in combination, TNF-alpha and IFN-gamma synergistically stimulated the secretion of both laminin-5 and -10 in HIEC cells. Transcript analyses suggested that the upregulation of the two laminins might depend on distinct mechanisms. Interestingly, the TNF-alpha and IFN-gamma combination was also found to significantly promote apoptosis. However, the effect of cytokines on the secretion of laminins was maintained even after completely blocking apoptosis by inhibiting caspase activities. These results demonstrate that laminin production is specifically modulated by the proinflammatory cytokines TNF-alpha and IFN-gamma in intestinal epithelial cells under an apoptosis-independent mechanism.