Ciro Abbondanza

Steroid‐induced androgen receptor–oestradiol receptor β–Src complex triggers prostate cancer cell proliferation

Treatment of human prostate carcinoma‐derived LNCaP cells with androgen or oestradiol triggers simultaneous association of androgen receptor and oestradiol receptor β with Src, activates the Src/Raf‐1/Erk‐2 pathway and stimulates cell proliferation. Surprisingly, either androgen or oestradiol action on each of these steps is inhibited by both anti‐androgens and anti‐oestrogens. Similar findings for oestradiol receptor α were observed in MCF‐7 or T47D cells stimulated by either oestradiol or androgens. Microinjection of LNCaP, MCF‐7 and T47D cells with SrcK− abolishes steroid‐stimulated S‐phase entry. Data from transfected Cos cells confirm and extend the findings from these cells. Hormone‐stimulated Src interaction with the androgen receptor and oestradiol receptor α or β is detected using glutathione S‐transferase fusion constructs. Src SH2 interacts with phosphotyrosine 537 of oestradiol receptor α and the Src SH3 domain with a proline‐rich stretch of the androgen receptor. The role of this phosphotyrosine is stressed by its requirement for association of oestradiol receptor α with Src and consequent activation of Src in intact Cos cells.

DNA Oxidation as Triggered by H3K9me2 Demethylation Drives Estrogen-Induced Gene Expression

Modifications at the N-terminal tails of nucleosomal histones are required for efficient transcription in vivo. We analyzed how H3 histone methylation and demethylation control expression of estrogen-responsive genes and show that a DNA-bound estrogen receptor directs transcription by participating in bending chromatin to contact the RNA polymerase II recruited to the promoter. This process is driven by receptor-targeted demethylation of H3 lysine 9 at both enhancer and promoter sites and is achieved by activation of resident LSD1 demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine–DNA glycosylase 1 and topoisomeraseIIβ, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Our data show a strategy that uses controlled DNA damage and repair to guide productive transcription.

17beta-estradiol inhibits apoptosis in MCF-7 cells, inducing bcl-2 expression via two estrogen-responsive elements present in the coding sequence.

ABSTRACT We have found that 17β-estradiol induces bcl-2transcription in human breast cancer MCF-7 cells. To identifycis-acting elements involved in this regulation, we have analyzed hormone responsiveness of transiently transfected reporter constructs containing the bcl-2 major promoter (P1). Hormone inducibility was observed only when either of two sequences, located within the bcl-2 coding region and showing one and two mutations with respect to the consensus estrogen-responsive element, were inserted downstream from the P1 promoter. Both sequences behaved as enhancers exclusively in cells expressing the estrogen receptor and were able to bind this receptor in in vitro assays. Transfections into MCF-7 cells of plasmids carrying a bcl-2 cDNA fragment which included these two elements revealed that their simultaneous presence resulted in an additive effect on reporter gene activity, whose size resembled the increase of endogenous bcl-2 mRNA level observed in untransfected cells after hormone treatment. Moreover, the identified elements were able to mediate up-regulation ofbcl-2 expression by 17β-estradiol, since exogenousbcl-2 mRNA was induced by hormone challenge of MCF-7 cells transiently transfected with a vector containing the bcl-2coding sequence cloned under the control of a non-estrogen-responsive promoter. Finally, we show that hormone prevention of apoptosis, induced by incubating MCF-7 cells with hydrogen peroxide, was strictly related to bcl-2 up-regulation. Our results indicate that the bcl-2 major promoter does not containcis-acting elements directly involved in transcriptional control by 17β-estradiol and that hormone treatment inhibits programmed cell death in MCF-7 cells, inducing bcl-2expression via two estrogen-responsive elements located within its coding region.

γ1- and γ2-Syntrophins, Two Novel Dystrophin-binding Proteins Localized in Neuronal Cells

Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies. At the last 3′ terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound. These are a family of cytoplasmic peripheral membrane proteins. Three genes have been described encoding one acidic (α1) and two basic (β1 and β2) proteins of ∼57–60 kDa. Here, we describe the characterization of two novel putative members of the syntrophin family, named γ1- and γ2-syntrophins. The human γ1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids. The human γ2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues. We mapped the γ1-syntrophin gene to human chromosome 8q11 and the γ2-syntrophin gene to chromosome 2p25. Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins. We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections. Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system.

Characterization and epitope mapping of a new panel of monoclonal antibodies to estradiol receptor

A new panel of monoclonal antibodies to the calf uterus estrogen receptor was prepared. Thirteen antibodies were characterized for their isotype and for the affinity for the antigen. These antibodies recognize the human receptor and can be used in Western blot analysis. The location of the epitopes was mapped on the antigen structure using synthetic fragments of estrogen receptor, and it was possible to group the antibodies in five groups. Many antibodies were useful for the purification of estrogen receptor from tissue extracts by immunoaffinity chromatography. The reciprocal inhibition of the antibodies for the antigen binding was measured with an immunoadsorption assay. This was maximal and symmetrical for antibody pairs within the same group, but was incomplete and, in some instances, asymmetrical between pairs of antibodies from different groups. One antibody was able to inhibit the estrogen receptor-DNA interaction, whereas two others were unable to recognize the receptor-DNA complexes. This new panel of antibodies is a useful addition to the existing tools for studying structure and function of the estrogen receptor.

Tyrosine phosphorylation of estradiol receptor by Src regulates its hormone-dependent nuclear export and cell cycle progression in breast cancer cells

We report that in breast cancer cells, tyrosine phosphorylation of the estradiol receptor alpha (ERalpha) by Src regulates cytoplasmic localization of the receptor and DNA synthesis. Inhibition of Src or use of a peptide mimicking the ERalpha p-Tyr537 sequence abolishes ERalpha tyrosine phosphorylation and traps the receptor in nuclei of estradiol-treated MCF-7 cells. An ERalpha mutant carrying a mutation of Tyr537 to phenylalanine (ER537F) persistently localizes in nuclei of various cell types. In contrast with ERalpha wt, ER537F does not associate with Ran and its interaction with Crm1 is insensitive to estradiol. Thus, independently of estradiol, ER537F is retained in nuclei, where it entangles FKHR-driving cell cycle arrest. Chromatin immunoprecipitation analysis reveals that overexpression of ER537F in breast cancer cells enhances FKHR interaction with cyclin D1 promoter. This mutant also counteracts cell transformation by the activated forms of Src or PI3-K. In conclusion, in addition to regulating receptor localization, ERalpha phosphorylation by Src is required for hormone responsiveness of DNA synthesis in breast cancer cells.

Raloxifene induces cell death and inhibits proliferation through multiple signaling pathways in prostate cancer cells expressing different levels of estrogen receptorα and β

Raloxifene (RAL), a selective estrogen receptor (ER) modulator (SERM) seems to induce apoptosis in both androgen‐dependent and ‐independent prostate cell (PC) lines via activation of ERβ and an antagonistic effect on ERα. In this study, we evaluated the effects of RAL on epithelial PC growth using the two following in vitro models: the androgen‐dependent cell line EPN which expressed both ERs; and a stabilized epithelial cell line derived from a prostate cancer specimen (CPEC), which expressed low levels of ERβ and lacked ERα. In EPN cells, there was an increase in the pre‐G1 apoptotic peak and a reduction in the S phase of the cell cycle with G0/G1 arrest after E2 or RAL treatment; bcl‐2 mRNA and Bcl‐2 protein levels were significantly reduced, while activated caspase‐3 and Par‐4 levels increased significantly after either E2 or RAL treatment; in addition, c‐myc transcript was inhibited after 10−6 M RAL treatment. A dose‐dependent increase of metallothionein II gene RNA level was also induced by RAL in EPN. In CPEC, there was only a weak apoptotic peak associated with caspase‐3 activation and Par‐4 increase after either E2 or RAL treatment; while c‐myc transcript level increased. RAL induced a rapid but transient phosphorylation of ERK 1/2 in EPN cells but generated a sustained effect in CPEC. These findings suggest that RAL effects on PC growth control in vitro are cell‐specific, depending on ERβ or ERβ/ERα relative expression levels. Moreover, this study demonstrated that RAL affected both transcriptional regulation and non‐genomic signals, which resulted in the modulation of multiple signaling pathways of apoptosis and of cell cycle progression. J. Cell. Physiol. 226: 1334–1339, 2011.

Kidney and heart interactions during cardiorenal syndrome: a molecular and clinical pathogenic framework.

The heart and kidney are physiologically interconnected. Cardiorenal syndrome (CRS) is a pathological disorder where acute or chronic dysfunction in one organ may induce dysfunction in the other one. Although classical studies have proposed a role for hypertension, dyslipidemia and endothelial dysfunction, CRS should be considered as a complex molecular interplay of neurohumoral pathway activation including the sympathetic nervous system, the renin angiotensin aldosterone axis, the endothelin system and the arginine vasopressin system. This activation may induce vascular inflammation, oxidative stress, accelerated atherosclerosis, cardiac hypertrophy and both myocardial and intrarenal fibrosis with progression of CRS treatment. More recently, epigenetics has opened new pathogenic molecular routes for CRS. This will lead to a more rapid development of novel, safe and effective clinical therapies.

Detrimental effects of Bartonella henselae are counteracted by L-arginine and nitric oxide in human endothelial progenitor cells.

The recruitment of circulating endothelial progenitor cells (EPCs) might have a beneficial effect on the clinical course of several diseases. Endothelial damage and detachment of endothelial cells are known to occur in infection, tissue ischemia, and sepsis. These detrimental effects in EPCs are unknown. Here we elucidated whether human EPCs internalize Bartonella henselae constituting a circulating niche of the pathogen. B. henselae invades EPCs as shown by gentamicin protection assays and transmission electron microscopy (TEM). Dil-Ac-LDL/lectin double immunostaining and fluorescence-activated cell sorting (FACS) analysis of EPCs revealed EPC bioactivity after infection with B. henselae. Nitric oxide (NO) and its precursor l-arginine (l-arg) exert a plethora of beneficial effects on vascular function and modulation of immune response. Therefore, we tested also the hypothesis that l-arg (1–30 mM) would affect the infection of B. henselae or tumor necrosis factor (TNF) in EPCs. Our data provide evidence that l-arg counteracts detrimental effects induced by TNF or Bartonella infections via NO (confirmed by DETA-NO and L-NMMA experiments) and by modulation of p38 kinase phosphorylation. Microarray analysis indicated several genes involved in immune response were differentially expressed in Bartonella-infected EPCs, whereas these genes returned in steady state when cells were exposed to sustained doses of l-arg. This mechanism may have broad therapeutic applications in tissue ischemia, angiogenesis, immune response, and sepsis.

Prostate cancer stem cells: the role of androgen and estrogen receptors

Prostate cancer is one of the most commonly diagnosed cancers in men, and androgen deprivation therapy still represents the primary treatment for prostate cancer patients. This approach, however, frequently fails and patients develop castration-resistant prostate cancer, which is almost untreatable. Cancer cells are characterized by a hierarchical organization, and stem/progenitor cells are endowed with tumor-initiating activity. Accumulating evidence indicates that prostate cancer stem cells lack the androgen receptor and are, indeed, resistant to androgen deprivation therapy. In contrast, these cells express classical (α and/or β) and novel (GPR30) estrogen receptors, which may represent new putative targets in prostate cancer treatment. In the present review, we discuss the still-debated mechanisms, both genomic and non-genomic, by which androgen and estradiol receptors (classical and novel) mediate the hormonal control of prostate cell stemness, transformation, and the continued growth of prostate cancer. Recent preclinical and clinical findings obtained using new androgen receptor antagonists, anti-estrogens, or compounds such as enhancers of androgen receptor degradation and peptides inhibiting non-genomic androgen functions are also presented. These new drugs will likely lead to significant advances in prostate cancer therapy.