Transcription factor Fra-2 and its emerging role in matrix deposition, proliferation and inflammation in chronic lung diseases.

Fos-related antigen-2 (Fra-2) belongs to the activator protein 1 (AP-1) family of transcription factors and is involved in a broad variety of cellular processes, such as proliferation or differentiation. Aberrant expression of Fra-2 or regulation can lead to severe growth defects or diverse pathologies. Elevated Fra-2 expression has been described in several chronic lung diseases, such as pulmonary fibrosis, chronic obstructive pulmonary disease and asthma. However, the pathomechanisms behind the Fra-2-induced pulmonary remodelling are still not fully elucidated. Fra-2 overexpressing mice were initially described as a model of systemic sclerosis associated organ fibrosis, with predominant alterations in the lung. High levels of Fra-2 expression give rise to profound inflammation with severe remodelling of the parenchyma and the vasculature, resulting in fibrosis and pulmonary hypertension, respectively, but also alters bronchial function. In this review we discuss the central role of Fra-2 connecting inflammation, cellular proliferation and extracellular matrix deposition underlying chronic lung diseases and what we can learn for future therapeutic options.

Fos-like antigen 2 (FOSL2) promotes metastasis in colon cancer.

Among different cancers, incidence and mortality of colorectal cancer (CRC) is one of the highest. KRAS mutation is one of the underlying features in the pathogenesis of CRC with CRC tumors harboring mutant KRAS exhibiting a more aggressive behavior compared to CRC tumors with wild type KRAS. We had earlier shown that the microRNA-143 (miR-143) replenishment not only chemosensitizers CRC cell line with mutant KRAS instead of wild-type KRAS gene, to paclitaxel-mediated cytotoxicity, but also inhibits cell migration and invasion ability. Hence, the study aimed to determine how miR-143 replenishment is inhibiting pre-metastatic behavior in CRC cells with mutant KRAS. Top ten mRNA targets of miR-143 as predicted by TargetScan were evaluated by qRT-PCR in LoVo cells which were performed mock transfection or miR-143 mimic transfection. Evaluation of the changes in cognate mRNA target(s) was done in 30 paired CRC tissue and tumor adjacent normal tissue specimens and in LoVo cells by western blot. Effect of the mRNA target on pro-metastatic behavior was assayed by gain- and loss-of-function studies using a combination of western blotting and in vitro cell proliferation and transwell migration/invasion assay in LoVo cells and in the normal colonic epithelium cell line FHC. In vivo effect of the cognate mRNA target on CRC metastasis was assayed by xenograft assay. Of the 10 predicted mRNA targets, FOSL2 (P < 0.05) and IGFBP5 (P > 0.05) was down regulated in LoVo cells transfected with the miR-143 mimic. FOSL2 mRNA levels were significantly downregulated in CRC tissue specimens compared with adjacent normal tissue (P < 0.05). Immunoblot analysis showed that FOSL2, but not IGFBP5, protein expression is down regulated in LoVo cells after the miR-143 mimic transfection. FOSL2 overexpression in the normal colonic epithelial cell line FHC or siRNA-mediated silencing in LoVo cells induced and repressed, respectively, pro-mesenchymal cell features. Whereas manipulation of FOSL2 expression did not have any effect on cell proliferation rates, silencing its expression inhibited cell migration and invasion ability in vitro. In addition, silencing of FOSL2 expression in the LoVo cells can significantly inhibited invasion of hepatic, while no effect was found for tumorigenic potential. Our results suggest that FOSL2 is a critical regulator of CRC metastasis and might be an important marker for prognostic in CRC patients.

Histone Demethylation and Toll-like Receptor 8-Dependent Cross-Talk in Monocytes Promotes Transdifferentiation of Fibroblasts in Systemic Sclerosis Via Fra-2.

OBJECTIVE: To investigate whether epigenetic changes can modulate monocytes to produce tissue inhibitor of metalloproteinases 1 (TIMP-1) via Fra-2 (an activator protein 1 [AP-1] family member), a novel downstream mediator that promotes fibrogenesis. METHODS: AP-1 transcription factors and TIMP-1 expression were measured in monocytes from systemic sclerosis (SSc) patients and healthy controls. Involvement of Fra-2 in the regulation of TIMP-1 following treatment with Toll-like receptor 8 (TLR-8) agonist was investigated using a luciferase activity assay and chromatin immunoprecipitation (ChIP) analysis. Expression of TIMP-1 and Fra-2 was determined in response to TLR-8 treatment and to different histone modifications, including 3'-deazaneplanocin (DZNep) and apicidin. Fibroblasts from healthy controls were cocultured with DZNep plus TLR-8-treated healthy control monocytes. RESULTS: Up-regulation of Fra-2 was detected in bleomycin-challenged mice and in skin biopsy samples from SSc patients. Enhanced expression of Fra-2 and TIMP-1 was correlated in SSc monocytes (P = 0.021). The expression of Fra-1 was significantly reduced (P = 0.037) in SSc monocytes. Inhibiting AP-1 activity reduced TIMP-1 production in TLR-8-stimulated monocytes from healthy controls and SSc patients. ChIP experiments revealed binding of Fra-2 to the TIMP-1 promoter. Stimulation with DZNep plus TLR-8 enhanced Fra-2 and TIMP-1 expression in healthy control monocytes, whereas TLR-8 plus apicidin repressed Fra-2 and TIMP-1 expression. Finally, healthy control monocytes treated with DZNep plus TLR-8 induced strong production of α-smooth muscle actin in dermal fibroblasts, which was inhibited by TIMP-1-blocking antibody. CONCLUSION: These data demonstrate a novel role of histone demethylation induced by DZNep on Fra-2-mediated TIMP-1 production by monocytes in the presence of TLR-8 agonist. This consequently orchestrates the transdifferentiation of fibroblasts, a key event in the pathogenesis of SSc.

SOX4 is a direct target gene of FRA-2 and induces expression of HDAC8 in adult T-cell leukemia/lymphoma.

Previously, we have shown that an AP-1 family member, FRA-2, is constitutively expressed in adult T-cell leukemia/lymphoma (ATL) and, together with JUND, upregulates CCR4 and promotes ATL cell growth. Among the identified potential target genes of FRA-2/JUND was SOX4. Here, we examine the expression and function of SOX4 in ATL. SOX4 was indeed consistently expressed in primary ATL cells. FRA-2/JUND efficiently activated the SOX4 promoter via an AP-1 site. Knockdown of SOX4 expression by small interfering RNA (siRNA) strongly suppressed cell growth of ATL cell lines. Microarray analyses revealed that SOX4 knockdown reduced the expression of genes such as germinal center kinase related (GCKR), NAK-associated protein 1 (NAP1), and histone deacetylase 8 (HDAC8). We confirmed consistent expression of GCKR, NAP1, and HDAC8 in primary ATL cells. We also showed direct activation of the HDAC8 promoter by SOX4. Furthermore, siRNA knockdown of GCKR, NAP1, and HDAC8 each significantly suppressed cell growth of ATL cell lines. Taken together, we have revealed an important oncogenic cascade involving FRA-2/JUND and SOX4 in ATL, which leads to the expression of genes such as GCKR, NAP1, and HDAC8.

Mechanisms of acute uremic encephalopathy: early activation of Fos and Fra-2 gene products in different nuclei/areas of the rat brain.

High levels of various uremic toxins such as guanidino compounds and advanced glycation endproducts, as well as an excess of parathyroid hormones, are involved in the pathogenesis of acute uremic encephalopathy. Moreover, distant effects of the damaged kidney with enhanced production of inflammatory mediators are implicated. Data on the pump activity of an abnormal Na-K-ATPase and inhibition of the organic anion transporter system in the brain have been published previously. Recently, the effect of an experimentally induced acute renal failure (ARF) on the neuronal cell activation of Fos and Fra-2 in the rat brain was investigated by immunohistochemistry. ARF was induced by using the following 3 rat models: bilateral nephrectomy, bilateral ureter ligation, and uranyl acetate injection with corresponding controls. The Fos and the Fra-2 immunoreactive neurons of the brain were determined in a total of 120 brain areas over a period of 3 days post bilateral nephrectomy and bilateral ureter ligation and 12 days after uranyl acetate. An activation response was observed in 73 of 120 areas of the brain. The responses were classified into 4 groups: (1) biogenic amines (noradrenaline, adrenaline, histamine, and 5-hydroxytryptamine), (2) stress-sensitive forebrain areas, (3) neuronal cell groups involved in the regulation of water and electrolyte homeostasis, and (4) central autonomic cell groups. In the uranyl acetate-induced ARF, activation of Fos and Fra-2 immunoreactivity took place at the earliest time-point (3 hours) which persisted even after improvement of ARF. This suggests the involvement of the toxic effects of uranium as a result of its accumulation in the brain.

The transcription factor Fra-2 promotes mammary tumour progression by changing the adhesive properties of breast cancer cells.

The transcription factor Fra-2 (Fos-related antigen-2) has been implicated in invasion of breast cancer cells, but there is only sparse information about its role in clinical tumours. In the present study, we analysed Fra-2 mRNA expression in a cohort of 167 patients, and found significant correlations between high Fra-2 expression and nodal involvement or reduced disease-free survival. To get more information about the underlying mechanisms, we generated stably transfected MDA-MB231 breast cancer cells with increased Fra-2 expression. Compared with the controls, these clones did not differ in proliferation and motility, but had higher invasive potential. By global gene expression analysis and subsequent validation of selected genes, we identified a number of proteins involved in cell-cell or cell-matrix interactions that were up- or down-regulated in Fra-2 overexpressing cells, e.g. connexin 43, ICAM-1, L1-CAM, integrin beta 2, integrin beta 4, and integrin alpha 6. The association of Fra-2 overexpression and high ICAM-1 or L1-CAM levels could also be demonstrated in our clinical cohort of mammary tumours. In both MDA-MB231 and MCF7 cells, we found an increased attachment of Fra-2 transfectants to components of the extracellular matrix. In addition, we could show a striking increase in the number of rolling cells in flow-through assays using E-selectin coated capillaries, which might indicate a higher capacity of extravasation. In conclusion, our data obtained on breast cancer cell lines and clinical tissue samples suggest that overexpression of Fra-2 promotes breast cancer progression and metastasis by deregulation of genes involved in cell-cell and cell-ECM contacts.

Transcription factor fos-related antigen-2 induces progressive peripheral vasculopathy in mice closely resembling human systemic sclerosis.

BACKGROUND: Microvascular damage is one of the first pathological changes in systemic sclerosis. In this study, we investigated the role of Fos-related antigen-2 (Fra-2), a transcription factor of the activator protein-1 family, in the peripheral vasculopathy of systemic sclerosis and examined the underlying mechanisms. METHODS AND RESULTS: Expression of Fra-2 protein was significantly increased in skin biopsies of systemic sclerosis patients compared with healthy controls, especially in endothelial and vascular smooth muscle cells. Fra-2 transgenic mice developed a severe loss of small blood vessels in the skin that was paralleled by progressive skin fibrosis at 12 weeks of age. The reduction in capillary density was preceded by a significant increase in apoptosis in endothelial cells at week 9 as detected by immunohistochemistry. Similarly, suppression of Fra-2 by small interfering RNA prevented human microvascular endothelial cells from staurosporine-induced apoptosis and improved both the number of tubes and the cumulative tube lengths in the tube formation assay. In addition, cell migration in the scratch assay and vascular endothelial growth factor-dependent chemotaxis in a modified Boyden chamber assay were increased after transfection of human microvascular endothelial cells with Fra-2 small interfering RNA, whereas proliferation was not affected. CONCLUSIONS: Fra-2 is present in human systemic sclerosis and may contribute to the development of microvasculopathy by inducing endothelial cell apoptosis and by reducing endothelial cell migration and chemotaxis. Fra-2 transgenic mice are a promising preclinical model to study the mechanisms and therapeutic approaches of the peripheral vasculopathy in systemic sclerosis.

Aberrant selection and function of invariant NKT cells in the absence of AP-1 transcription factor Fra-2.

The transcription factors mediating the development of CD1d-restricted invariant NKT (iNKT) cells remain incompletely described. Here, we show that loss of the AP-1 transcription factor Fra-2 causes a marked increase in the number of both thymic and peripheral iNKT cells, without affecting the development of other T-lineage cells. The defect is cell-autonomous and is evident in the earliest iNKT precursors. We find that iNKT cells expressing the lower affinity TCRVbeta8 are proportionally overrepresented in the absence of Fra-2, indicating altered selection of iNKT cells. There is also widespread dysregulation of AP-1-directed gene expression. In the periphery, mature Fra-2-deficient iNKT cells are able to participate in an immune response, but they have an altered response to Ag, showing increased expansion and producing increased amounts of IL-2 and IL-4 compared with their wild-type counterparts. Unusually, naive Fra-2-deficient T cells also rapidly produce IL-2 and IL-4 upon activation. Taken together, these data define Fra-2 as necessary for regulation of normal iNKT cell development and function, and they demonstrate the central role played by the AP-1 family in this lineage.

Alterations in fos-related antigen 2 and sigma1 receptor gene and protein expression are associated with the development of cocaine-induced behavioral sensitization: time course and regional distribution studies.

Repeated exposure to cocaine results in neuroadaptations that can alter the way the brain responds to subsequent stimuli. Earlier studies demonstrated that acute administration of cocaine up-regulates the immediate-early gene fos-related antigen 2 (fra-2) followed by a later up-regulation of sigma(1) receptor gene and protein levels in brain regions involved in addiction and reward. To test whether such alterations could have long-term consequences on behavior, the present study was undertaken. Using a cocaine-induced behavioral sensitization model coupled with gene and protein expression studies in mice, the results show that cocaine induces the expression of fra-2, which leads to a progressive increase in sigma(1) receptor gene and protein expression over a period of days. This progressive increase in sigma(1) expression corresponds to the steady increase in the locomotor response to repeated cocaine administration in mice. The cocaine-induced changes in fra-2 and sigma(1) receptor gene and protein expression occur in brain regions that subserve drug abuse, such as the cortex, striatum, and hippocampus, but not the cerebellum. Moreover, the prototypic sigma(1) receptor antagonist 1-[2-(3,4-dichloropheny)ethyl]-4-methylpiperazine (BD1063) significantly attenuates both the molecular adaptations and behavioral sensitization induced by cocaine. These data suggest that repeated exposure to cocaine elicits alterations in fra-2 and sigma(1) receptor-mediated mechanisms, which ultimately manifest as altered behavioral responses to cocaine.

Expression of AP-1 family transcription factors in the amygdala during conditioned taste aversion learning: role for Fra-2.

Conditioned taste aversion (CTA) learning occurs after the pairing of a novel taste with a toxin (e.g. sucrose with LiCl). The immediate early gene c-Fos is necessary for CTA learning, but c-Fos alone cannot be sufficient for consolidation. The expression of other AP-1 proteins from the Fos- and Jun-families may also be required shortly after conditioning for CTA consolidation. To screen for the expression of AP-1 transcription factors within small subregions, RT-PCR analysis was used after laser capture microdissection of the amygdala. Rats were infused intraorally with 5% sucrose (6 ml/6 min) or injected with LiCl (12 ml/kg, 0.15 M, i.p.) or given sucrose paired with LiCl (sucrose/LiCl), or not treated; 1 h later their brains were dissected. The lateral (LA), basolateral (BLA), and central (CeA) subnuclei of the amgydala of single 5 microm sections from individual rats were dissected using the Arcturus PixCell II system. Semi-quantitative RT-PCR showed the consistent presence of c-Fos, Fra-2, c-Jun, and JunD in the amygdala. In situ hybridization confirmed that c-Fos and Fra-2 mRNA expression was increased in the CeA after LiCl and sucrose/LiCl treatment. Immunohistochemistry for Fra-2 revealed high baseline levels of Fra-2 protein in the BLA and CeA, but also an increase in Fra-2 in the BLA and CeA after LiCl and sucrose/LiCl treatment. The similarity of response in LiCl and sucrose/LiCl treated groups might reflect activation by LiCl in both groups. To control for the effects of LiCl, rats were tested in a learned safety experiment. Fra-2 and c-Fos were examined in response to sucrose/LiCl in rats with prior familiarity with sucrose compared to rats without prior exposure to sucrose. The familiar (pre-exposure) group showed a significantly decreased number of Fra-2-positive cells compared with the novel group in the BLA, but not in the CeA. Because pre-exposure to sucrose attenuates CTA learning, a decreased cellular response in pre-exposed rats suggests a specific correlation with CTA learning. Changes in Fra-2 and c-Fos expression in the BLA and CeA at the time of conditioning, together with constitutive expression of c-Jun and JunD, may contribute to CTA learning.

[CCR4, HTLV-1 infection, and ATL oncogenesis].

Adult T-cell leukemia (ATL) is a malignancy of mature CD4+ T cells that is etiologically associated with the infection of human T-cell leukemia virus type 1 (HTLV-1), an exogenous human retrovirus. Previously, we have shown that leukemic cells of most ATL patients express CCR4, a chemokine receptor known to be selectively expressed by T cell subsets such as Th2 cells, skin-homing memory/effector T cells, and regulatory T cells. Therefore, the expression of CCR4 suggests that ATL cells are mostly derived from one of these T cell subsets. We have also shown that Tax, the HTLV-1-encoded potent transcriptional activator, strongly induces the expression of CCL22, a CCR4 ligand, which promotes the cell-dependent transmission of HTLV-1 from HTLV-1-infected T cells to CCR4+ target T cells by inducing close cell-to-cell interactions. We have also shown that ATL cells aberrantly express the AP-1 family member Fra-2 which, by forming the heterodimer with JunD, potently induces the expression of not only CCR4 but also the genes such as c-Myb, MDM2 and Bcl-6, the well-known proto-oncogenes. Thus, Fra-2 is a novel oncogene of ATL, and CCR4 may be regarded as a useful tumor marker of ATL.

Role of Fra-2 in breast cancer: influence on tumor cell invasion and motility.

Fra-2 (Fos-related antigen 2) is a member of the Fos family of AP-1 transcription factors which is often up-regulated in mammary carcinomas. Previous results suggested that it might be involved in the regulation of breast cancer invasion and metastasis. In order to analyze the role of Fra-2 in breast cancer cells, it was silenced in the highly invasive MDA-MB231 cells using RNA interference. On the other hand, stable transfectants of the weakly invasive MCF7 cell line were established in order to analyze the effects of Fra-2 overexpression. In both approaches, cell proliferation was not or only weakly influenced by Fra-2. In contrast, the invasive potential of the cells was increased, and a weaker effect on motility was observed. By cDNA microarray analysis of the MCF7 transfectants followed by validation on a protein level, we identified several Fra-2 target genes which might be involved in cell invasion and migration, i.e., ALCAM and connexin 43. Additionally, mRNA expression levels of various genes which are associated with a more malignant behavior of the tumors in vivo were up- or downregulated, i.e., members of the MAGE family, S100P, TIMP2, IL24 etc. These results show that Fra-2 overexpression is associated with a more aggressive tumor phenotype and is probably involved in breast cancer progression in vivo.

The role of repressor proteins in the adrenergic induction of type II iodothyronine deiodinase in rat pinealocytes.

In this study, we investigated the transcriptional regulation of the adrenergic induction of type II iodothyronine deiodinase (Dio2) in rat pinealocytes. Treatment of pinealocytes with norepinephrine (NE) caused an increase in the mRNA level of Dio2 that peaked around 2 h and declined over the next 5 h. Both beta- and alpha1-adrenergic receptors contributed to the NE induction of Dio2 expression through a cAMP/protein kinase A mechanism. In pinealocytes that had been stimulated by NE, inhibition of transcription by actinomycin had no discernible effect on Dio2 expression. In contrast, inhibition of protein synthesis by cycloheximide enhanced the NE induction of Dio2 expression, suggesting the involvement of a repressor protein. Transient transfection of pinealocytes with adenovirus expressing small interfering RNA against Fos-related antigen 2 (Fra2) enhanced the NE induction of Dio2 expression, whereas the effect of overexpression of the full-length transcript of Fra2 was inhibitory. Time-course study indicated that preventing the NE induction of Fra2 enhanced the NE induction of Dio2 after 3 h, and the enhancement persisted beyond 6 h after NE stimulation. In comparison, transient transfection of pinealocytes with small interfering RNA against inducible cAMP early repressor (Icer) had no effect on the NE induction of Dio2 expression, whereas overexpression of the full-length transcript of Icer caused a small reduction of the NE-stimulated Dio2 expression. Together, our results support Fra-2 as an important transcriptional repressor that helps shape the time profile of the adrenergic induction of Dio2 expression in the rat pineal gland.

The role of inducible repressor proteins in the adrenergic induction of arylalkylamine-N-acetyltransferase and mitogen-activated protein kinase phosphatase-1 in rat pinealocytes.

In this study, we investigated the role of two inducible repressor proteins, inducible cAMP early repressor (ICER) and Fos-related antigen 2 (Fra-2) in the adrenergic induction of MAPK phosphatase-1 (MKP-1) as compared with their roles in the induction of arylalkylamine-N-acetyltransferase (AA-NAT) in rat pinealocytes. Treatment of pinealocytes with norepinephrine (NE) caused an increase in the mRNA and protein levels of MKP-1 and AA-NAT, as well as in the AA-NAT activity and melatonin production. NE stimulation also caused a simultaneous increase in the mRNA and protein levels of ICER and Fra-2. Transient knockdown of icer using adenovirus expressing small interfering RNA (siRNA) abolished the NE induction of icer expression but had little effect on the NE induction of mkp-1 or aa-nat expression. In contrast, pretreatment with adenovirus overexpressing icer was effective in reducing the NE induction of mkp-1 and aa-nat. The inhibitory effect of overexpressing icer was reversed by cotreatment with siRNA against icer. siRNA against fra-2 also abolished the NE-stimulated expression of fra-2 but had little effect on the NE induction of mkp-1 and aa-nat expression. Proteasomal inhibition, which reduced the NE-stimulated induction of aa-nat, caused a reduction of ICER and Fra-2. Together, these results indicate that whereas overexpression of ICER can suppress the NE induction of aa-nat and mkp-1, the amount of the repressors, ICER and Fra-2, present during NE induction appears insufficient to exert a significant effect in controlling the expression of these genes.

Transcriptional repression of ATP-binding cassette transporter A1 gene in macrophages: a novel atherosclerotic effect of angiotensin II.

Angiotensin II (Ang II) is a powerful accelerator of atherosclerosis. Herein, we describe a novel transcription mechanism through which Ang II inhibits macrophage expression of the ATP-binding cassette transporter A1 (ABCA1), a key regulator of reverse cholesterol transport. We demonstrate that chronic Ang II infusion substantially promotes macrophage infiltration, foam cell formation, and atherosclerosis in low-density lipoprotein receptor-deficient mice and significantly reduces ABCA1 expression in peripheral macrophages. Administration of the Ang II type 1 receptor blocker valsartan inhibited Ang II-induced ABCA1 mRNA repression, macrophage cholesterol accumulation, and atherosclerosis. Ang II treatment reduced ABCA1 promoter activity of in vitro cultured mouse peritoneal macrophages, inducing fos-related antigen 2 (Fra2) protein binding to an ABCA1 promoter E-box motif, a site known to negatively regulate macrophage ABCA1 transcription. Valsartan pretreatment blocked Fra2 binding to the ABCA1 promoter, and Fra2 small interfering RNA pretreatment attenuated Ang II-mediated ABCA1 transcriptional inhibition, confirming the role of Fra2 in this process. This new evidence suggests that Ang II, a well-known proinflammatory and pro-oxidative factor, alters macrophage cholesterol homeostasis by repressing ABCA1 to promote foam cell formation and atherosclerosis.