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Epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) control the splicing pattern during epithelial to mesenchymal transition (EMT) in a physiological context and in cancer, including breast cancer (BC). Here, we report that ESRP1, but not ESRP2, is overexpressed in luminal BCs of patients with poor prognosis and correlates with estrogen receptor α (ERα) levels. Analysis of ERα genome-binding profiles in cell lines and primary breast tumors showed its binding in the proximity of ESRP1 and ESRP2 genes, whose expression is strongly decreased by ERα silencing in hormone-deprived conditions. The combined knock-down of ESRP1/2 in MCF-7 cells followed by RNA-Seq, revealed the dysregulation of 754 genes, with a widespread alteration of alternative splicing events (ASEs) of genes involved in cell signaling, metabolism, cell growth, and EMT. Functional network analysis of ASEs correlated with ESRP1/2 expression in ERα+ BCs showed RAC1 as the hub node in the protein–protein interactions altered by ESRP1/2 silencing. The comparison of ERα- and ESRP-modulated ASEs revealed 63 commonly regulated events, including 27 detected in primary BCs and endocrine-resistant cell lines. Our data support a functional implication of the ERα-ESRP1/2 axis in the onset and progression of BC by controlling the splicing patterns of related genes.

Simple Summary Aberrant alternative splicing is now considered a hallmark of cancer, including breast cancer. This results in the production of novel tumor-specific splice RNA variants, and the activation of biological processes such as epithelial-to-mesenchymal transition, leading to more aggressive phenotypes. The purpose of this study was to explore the role of estrogen receptor α in regulating the expression of RNA-binding proteins in luminal breast cancer cells and to determine the effects of its downregulation at the isoform level by exploring changes in isoform usage and alternative splicing. The findings of this study unravel a novel layer of gene regulation mediated by estrogen receptor α, which is fundamental for breast cancer cell growth as well as epithelial-to-mesenchymal transition. Finally, we foresee that this novel feature should be considered when studying the functional roles of estrogen receptor α in the onset and progression of breast cancer. Abstract Background: The transcriptional activity of estrogen receptor α (ERα) in breast cancer (BC) is extensively characterized. Our group has previously shown that ERα controls the expression of a number of genes in its unliganded form (apoERα), among which a large group of RNA-binding proteins (RBPs) encode genes, suggesting its role in the control of co- and post-transcriptional events. Methods: apoERα-mediated RNA processing events were characterized by the analysis of transcript usage and alternative splicing changes in an RNA-sequencing dataset from MCF-7 cells after siRNA-induced ERα downregulation. Results: ApoERα depletion induced an expression change of 681 RBPs, including 84 splicing factors involved in translation, ribonucleoprotein complex assembly, and 3′end processing. ApoERα depletion results in 758 isoform switching events with effects on 3′end length and the splicing of alternative cassette exons. The functional enrichment of these events shows that post-transcriptional regulation is part of the mechanisms by which apoERα controls epithelial-to-mesenchymal transition and BC cell proliferation. In primary BCs, the inclusion levels of the experimentally identified alternatively spliced exons are associated with overall and disease-free survival. Conclusion: Our data supports the role of apoERα in maintaining the luminal phenotype of BC cells by extensively regulating gene expression at the alternative splicing level.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects both lower and upper motor neurons (MNs) in the central nervous system. ALS etiology is highly multifactorial and multifarious, and an effective treatment is still lacking. Neuroinflammation is a hallmark of ALS and could be targeted to develop new therapeutic approaches. Interestingly, the transcription factor Nurr1 has been demonstrated to have an important role in the inflammatory process in several neurological disorders, such as Parkinson's disease and multiple sclerosis. In the present paper, we demonstrate for the first time that Nurr1 expression levels are upregulated in the peripheral blood of ALS patients. Moreover, we investigated Nurr1 function in the SOD1-G93A mouse model of ALS. Nurr1 was strongly upregulated in the spinal cord during the asymptomatic and early symptomatic phases of the disease, where it promoted the expression of brain-derived neurotrophic factor mRNA and the repression of NFκB pro-inflammatory targets, such as inducible nitric oxide synthase. Therefore, we hypothesize that Nurr1 is activated in an early phase of the disease as a protective endogenous anti-inflammatory mechanism, although not sufficient to reverse disease progression. On the basis of these observations, Nurr1 could represent a potential biomarker for ALS and a promising target for future therapies., Summary: We hypothesize that the transcription factor Nurr1 is activated in the early phase of the neurodegenerative disease amylotrophic lateral sclerosis (ALS), probably as a neuroprotective endogenous mechanism. Nurr1 might represent a promising target for ALS therapy.

Multiple Sclerosis (MS) is a chronic and potentially highly disabling disease that can cause permanent damage and deterioration of the central nervous system. In Europe it is the leading cause of non-traumatic disabilities in young adults, since more than 700,000 EU people suffer from MS. Although recent studies on MS pathophysiology have been provided, MS remains a challenging disease. In this context, thanks to recent advances in software and hardware technologies, computational models and computer simulations are becoming appealing research tools to support scientists in the study of such disease. Thus, motivated by this consideration we propose in this paper a new model to study the evolution of MS in silico, and the effects of the administration of Daclizumab drug, taking into account also spatiality and temporality of the involved phenomena. Moreover, we show how the intrinsic symmetries of the system can be exploited to drastically reduce the complexity of its analysis., Comment: Submitted to CIBB 2019 post proceedings - LNCS

Increasing evidence supports the anti-inflammatory role of estrogens in Multiple Sclerosis (MS), originating from the observation of reduction in relapse rates among women with MS during pregnancy, but the molecular mechanisms are still not completely understood. Using an integrative data analysis, we identified T helper (Th) 17 and T regulatory (Treg) cell-type-specific regulatory regions (CSR) regulated by estrogen receptor alpha (ERα). These CSRs were validated in polarized Th17 from healthy donors (HD) and in peripheral blood mononuclear cells, Th17 and Treg cells from relapsing remitting (RR) MS patients and HD during pregnancy. 17β-estradiol induces active histone marks enrichment at Forkhead Box P3 (FOXP3)-CSRs and repressive histone marks enrichment at RAR related orphan receptor C (RORC)-CSRs in polarized Th17 cells. A disease-associated epigenetic profile was found in RRMS patients during pregnancy, suggesting a FOXP3 positive regulation and a RORC negative regulation in the third trimester of pregnancy. Altogether, these data indicate that estrogens act as immunomodulatory factors on the epigenomes of CD4+ T cells in RRMS; the identified CSRs may represent potential biomarkers for monitoring disease progression or new potential therapeutic targets.

Estrogen Receptor alpha (ERα) activation by estrogenic hormones induces luminal breast cancer cell proliferation. However, ERα plays also important hormone-independent functions to maintain breast tumor cells epithelial phenotype. We reported previously by RNA-Seq that in MCF-7 cells in absence of hormones ERα down-regulation changes the expression of several genes linked to cellular development, representing a specific subset of estrogen-induced genes. Here, we report regulation of long non-coding RNAs from the same experimental settings. A list of 133 Apo-ERα-Regulated lncRNAs (AER-lncRNAs) was identified and extensively characterized using published data from cancer cell lines and tumor tissues, or experiments on MCF-7 cells. For several features, we ran validation using cell cultures or fresh tumor biopsies. AER-lncRNAs represent a specific subset, only marginally overlapping estrogen-induced transcripts, whose expression is largely restricted to luminal cells and which is able to perfectly classify breast tumor subtypes. The most abundant AER-lncRNA, DSCAM-AS1, is expressed in ERα+ breast carcinoma, but not in pre-neoplastic lesions, and correlates inversely with EMT markers. Down-regulation of DSCAM-AS1 recapitulated, in part, the effect of silencing ERα, i.e. growth arrest and induction of EMT markers. In conclusion, we report an ERα-dependent lncRNA set representing a novel luminal signature in breast cancer cells.

Pancreatic Ductal Adenocarcinoma (PDA) is characterized by a complex tumor microenvironment that supports its progression, aggressiveness and resistance to therapies. The delicate interplay between cancer and immune cells creates the conditions for PDA development, particularly due to the functional suppression of T cell anti-tumor effector activity. However, some of the mechanisms involved in this process are still poorly understood. In this study, we analyze whether the functional and epigenetic profile of T cells that infiltrate PDA is modulated by the microenvironment, and in particular by tumor-associated macrophages (TAMs). CD4 and CD8 T cells obtained from mice orthotopically injected with syngeneic PDA cells, and untreated or treated with Trabectedin, a cytotoxic drug that specifically targets TAMs, were sorted and analyzed by flow cytometry and characterized for their epigenetic profile. Assessment of cytokine production and the epigenetic profile of genes coding for IL10, T-bet and PD1 revealed that T cells that infiltrated PDA displayed activated Il10 promoter and repressed T-bet activity, in agreement with their regulatory phenotype (IL10high/IFNγlow, PD1high). By contrast, in Trabectedin-treated mice, PDA-infiltrating T cells displayed repressed Il10 and Pdcd1 and activated T-bet promoter activity, in accordance with their anti-tumor effector phenotype (IL10low/IFNγhigh), indicating a key role of TAMs in orchestrating functions of PDA-infiltrating T cells by modulating their epigenetic profile towards a pro-tumoral phenotype. These results suggest the targeting of TAMs as an efficient strategy to obtain an appropriate T cell anti-tumor immune response and open new potential combinations for PDA treatment.

Neuroglobin is a brain globin with neuroprotective effects against ischemia and related pathological processes, acting as O2 sensor and antiapoptotic pathway transducer. Here, we survey data on neuroanatomical coexpression of transcription factors, epigenetic signature and predictive transcription factor binding sites at the neuroglobin gene locus to find hints of pathways to neuroglobin transcriptional regulation. These data provide a glimpse of how neuroglobin expression may translate into neuronal diversity and function, as well as disease.

Pharmacological resistance is a serious threat to the clinical success of hormone therapy for breast cancer. The antiproliferative response to antagonistic drugs such as tamoxifen (Tam) critically depends on the recruitment of NCoR/SMRT corepressors to estrogen receptor alpha (ERα) bound to estrogen target genes. Under certain circumstances, as demonstrated in the case of interleukin-1β (IL-1β) treatment, the protein Tab2 interacts with ERα/NCoR and causes dismissal of NCoR from these genes, leading to loss of the antiproliferative response. In Tam-resistant (TamR) ER-positive breast cancer cells, we observed that Tab2 presents a shift in mobility on sodium dodecyl sulfate--PAGE (SDS-PAGE) similar to that seen in MCF7 wt upon stimulation with IL-1β, suggesting constitutive activation. Accordingly, TamR treatment with Tab2-specific short interfering RNA, restored the antiproliferative response to Tam in these cells. As Tab2 is known to directly interact with the N-terminal domain of ERα, we synthesized a peptide composed of a 14-aa motif of this domain, which effectively competes with ERα/Tab2 interaction in pull-down and co-immunoprecipitation experiments, fused to the carrier TAT peptide to allow internalization. Treatment of TamR cells with this peptide resulted in partial recovery of the antiproliferative response to Tam, suggesting a strategy to revert pharmacological resistance in breast cancer. Silencing of Tab2 in TamR cells by siRNA caused modulation of a gene set related to the control of cell cycle and extensively connected to BRCA1 in a functional network. These genes were able to discern two groups of patients, from a published data set of Tam-treated breast cancer profiles, with significantly different disease-free survival. Altogether, our data implicate Tab2 as a mediator of resistance to endocrine therapy and as a potential new target to reverse pharmacological resistance and potentiate antiestrogen action.

Diacylglycerol (DAG) kinases (Dgk), which phosphorylate DAG to generate phosphatidic acid, act as either positive or negative key regulators of cell signaling. We previously showed that Src mediates growth factors-induced activation of Dgk-alpha, whose activity is required for cell motility, proliferation and angiogenesis. Here, we demonstrate that both hepatocytes growth factor (HGF) stimulation and v-Src transformation induce tyrosine phosphorylation of Dgk-alpha on Y335, through a mechanism requiring its proline-rich C-terminal sequence. Moreover, we show that both proline-rich sequence and phosphorylation of Y335 of Dgk-alpha mediate: (i) its enzymatic activation, (ii) its ability to interact respectively with SH3 and SH2 domains of Src, (iii) its recruitment to the membrane. In addition, we show that phosphorylation of Dgk-alpha on Y335 is required for HGF-induced motility, while its constitutive recruitment at the membrane by myristylation is sufficient to trigger spontaneous motility in absence of HGF. Providing the first evidence that tyrosine phosphorylation of Dgk-alpha is required for growth-factors-induced activation and membrane recruitment, these findings underscore its relevance as a rheostat, whose activation is a threshold to elicit growth factors-induced migratory signaling.

Ghrelin is an acyl-peptide gastric hormone acting on the pituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, and appetite. Ghrelin endocrine activities are entirely dependent on its acylation and are mediated by GH secretagogue (GHS) receptor (GHSR)-1a, a G protein–coupled receptor mostly expressed in the pituitary and hypothalamus, previously identified as the receptor for a group of synthetic molecules featuring GH secretagogue (GHS) activity. Des-acyl ghrelin, which is far more abundant than ghrelin, does not bind GHSR-1a, is devoid of any endocrine activity, and its function is currently unknown. Ghrelin, which is expressed in heart, albeit at a much lower level than in the stomach, also exerts a cardio protective effect through an unknown mechanism, independent of GH release. Here we show that both ghrelin and des-acyl ghrelin inhibit apoptosis of primary adult and H9c2 cardiomyocytes and endothelial cells in vitro through activation of extracellular signal–regulated kinase-1/2 and Akt serine kinases. In addition, ghrelin and des-acyl ghrelin recognize common high affinity binding sites on H9c2 cardiomyocytes, which do not express GHSR-1a. Finally, both MK-0677 and hexarelin, a nonpeptidyl and a peptidyl synthetic GHS, respectively, recognize the common ghrelin and des-acyl ghrelin binding sites, inhibit cell death, and activate MAPK and Akt. These findings provide the first evidence that, independent of its acylation, ghrelin gene product may act as a survival factor directly on the cardiovascular system through binding to a novel, yet to be identified receptor, which is distinct from GHSR-1a.

Growth hormone secretagogues (GHSs) are synthetic peptidyl and nonpeptidyl molecules that possess strong growth hormone-releasing activity acting on specific pituitary and hypothalamic receptor subtypes. Differ- ently from nonpeptidyl GHSs, peptidyl molecules such as hexarelin, a hexapeptide, possess specific high-affin- ity binding sites in animal and human heart and, after prolonged treatment, protect rats in vivo from ischemia- induced myocardial damage. To verify the hypothesis that peptidyl GHSs protect heart cells from cell death, we have investigated the cellular effects of hexarelin on H9c2 cardiomyocytes, a fetal cardiomyocyte-derived cell line, and on Hend, an endothelial cell line derived from transformed murine heart endothelium. We show that (i)H9c2 cardiomyocytes show specific binding for 125I-Tyr-Ala-hexarelin, which is inhibited by peptidyl GHSs such as Tyr-Ala-hexarelin and hexarelin but not by the nonpeptidyl GHS MK-0677, (ii) hexarelin pro- motes survival of H9c2 cardiomyocytes induced to die by doxorubicin, and (iii) that hexarelin inhibits apoptosis, as measured by DNA fragmentation, in- duced in both H9c2 myocytes and endothelial cells. In conclusion, our findings show that peptidyl GHSs such as hexarelin act as survival factors for cardiomyocytes and endothelium-derived cells in culture. These find- ings suggest that the inhibitory activity of hexarelin on cardiomyocytes and endothelial cell death could explain, at least partially, its cardioprotective effect against ischemia recorded in rats in vivo.

Estrogen receptor-α (ERα) has central role in hormone-dependent breast cancer and its ligand-induced functions have been extensively characterized. However, evidence exists that ERα has functions that are independent of ligands. In the present work, we investigated the binding of ERα to chromatin in the absence of ligands and its functions on gene regulation. We demonstrated that in MCF7 breast cancer cells unliganded ERα binds to more than 4,000 chromatin sites. Unexpectedly, although almost entirely comprised in the larger group of estrogen-induced binding sites, we found that unliganded-ERα binding is specifically linked to genes with developmental functions, compared with estrogen-induced binding. Moreover, we found that siRNA-mediated down-regulation of ERα in absence of estrogen is accompanied by changes in the expression levels of hundreds of coding and noncoding RNAs. Down-regulated mRNAs showed enrichment in genes related to epithelial cell growth and development. Stable ERα down-regulation using shRNA, which caused cell growth arrest, was accompanied by increased H3K27me3 at ERα binding sites. Finally, we found that FOXA1 and AP2γ binding to several sites is decreased upon ERα silencing, suggesting that unliganded ERα participates, together with other factors, in the maintenance of the luminal-specific cistrome in breast cancer cells.

Motivations. In the last few years several studies showed that transposable elements (TEs) in the human genome are significantly associated with transcription factor binding sites (TFBSs) and that in several cases their expansion within the genome led to a substantial rewiring of the regulatory network. Here we suggest another possible role played by TEs in the evolution of the regulatory networks. We discuss a set of evidences supporting the idea that the evolution of particular patterns on combinatorial interactions among Transcription Factors (TFs) was mediated and supported by the expansion of specific classes of TEs in the human genome. Methods. We tested our conjecture looking, as a case study, at the binding of Estrogen Receptor alpha (ERalpha) to DNA using two chromatin immunoprecipitation sequencing (ChIP-seq) public datasets on MCF7 cell lines corresponding to different modalities of exposure to estrogen. On these datasets we performed a genome-wide analysis of Transposable Elements overlapping ChIP-seq binding peaks and for each of these sequences we performed a genome-wide scan for putative TFBSs employing canonical Positional Weight Matrices (PWMs) Results. We found a remarkable enrichment of a few well defined types and classes of transposable elements overlapping the ChiP-seq peaks in our two datasets. Among these enriched TEs a prominent role was played by MIR (Mammalian Interspersed Repeats) transposons. These TEs underwent a dramatic expansion at the beginning of the mammalian radiation and then stabilized. We conjecture that the special affinity of ERalpha for the MIR class of TEs could be at the origin of the important role which ERalpha assumed in mammalians. Then, looking at the results of the scan for TFBSs, we found strong enrichment and correlated presence of a few specific TFs. In several cases these TFs were known cofactors of ERalpha, thus supporting the idea of a co-regulatory role of Transcription Factors located within the sane TE. Most of these correlations turned out to be strictly associated to specific classes of TEs thus suggesting the presence of a well defined "transposon code" within the regulatory network. Altogether our results support the idea that transposition events, besides rewiring the network, also played a central role in the emergence and success of combinatorial gene regulation in complex eukaryotes and that the evolution of specific combinations of TFs interactions was actually mediated and driven by the expansion of a few specific classes of Transposable Elements.

Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. Inhere we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.

Diacylglycerol kinases (Dgk) phosphorylate diacylglycerol (DG) to phosphatidic acid (PA), thus turning off and on, respectively, DG-mediated and PA-mediated signaling pathways. We previously showed that hepatocyte growth factor (HGF), vascular endothelial growth factor, and anaplastic lymphoma kinase activate Dgkalpha in endothelial and leukemia cells through a Src-mediated mechanism and that activation of Dgkalpha is required for chemotactic, proliferative, and angiogenic signaling in vitro. Here, we investigate the downstream events and signaling pathways regulated by Dgkalpha, leading to cell scatter and migration upon HGF treatment and v-Src expression in epithelial cells. We report that specific inhibition of Dgkalpha, obtained either pharmacologically by R59949 treatment, or by expression of Dgkalpha dominant-negative mutant, or by small interfering RNA-mediated down-regulation of endogenous Dgkalpha, impairs 1) HGF- and v-Src-induced cell scatter and migration, without affecting the loss of intercellular adhesions; 2) HGF-induced cell spreading, lamellipodia formation, membrane ruffling, and focal adhesions remodeling; and 3) HGF-induced Rac activation and membrane targeting. In summary, we provide evidence that Dgkalpha, activated downstream of tyrosine kinase receptors and Src, regulates crucial steps directing Rac activation and Rac-dependent remodeling of actin cytoskeleton and focal contacts in migrating epithelial cells.

Vascular endothelial growth factor-A (VEGF-A) promotes angiogenesis by stimulating migration, proliferation and organization of endothelium, through the activation of signaling pathways involving Src tyrosine kinase. As we had previously shown that Src-mediated activation of diacylglycerol kinase-alpha (Dgk-alpha) is required for hepatocytes growth factor-stimulated cell migration, we asked whether Dgk-alpha is involved in the transduction of angiogenic signaling. In PAE-KDR cells, an endothelial-derived cell line expressing VEGFR-2, VEGF-A165, stimulates the enzymatic activity of Dgk-alpha: activation is inhibited by R59949, an isoform-specific Dgk inhibitor, and is dependent on Src tyrosine kinase, with which Dgk-alpha forms a complex. Conversely in HUVEC, VEGF-A165-induced activation of Dgk is only partially sensitive to R59949, suggesting that also other isoforms may be activated, albeit still dependent on Src tyrosine kinase. Specific inhibition of Dgk-alpha, obtained in both cells by R59949 and in PAE-KDR by expression of Dgk-alpha dominant-negative mutant, impairs VEGF-A165-dependent chemotaxis, proliferation and in vitro angiogenesis. In addition, in HUVEC, specific downregulation of Dgk-alpha by siRNA impairs in vitro angiogenesis on matrigel, further suggesting the requirement for Dgk-alpha in angiogenic signaling in HUVEC. Thus, we propose that activation of Dgk-alpha generates a signal essential for both proliferative and migratory response to VEGF-A165, suggesting that it may constitute a novel pharmacological target for angiogenesis control.

Activation of the receptor tyrosine kinase ErbB4 leads to various cellular responses such as proliferation, survival, differentiation, and chemotaxis. Two pairs of naturally occurring ErbB4 isoforms differing in their juxtamembrane (JMa/JMb) and C termini (cyt1/cyt2) have been described. To examine the role of ErbB4 in neuron migration, we cloned and stably transfected each of the four ErbB4 isoforms in ST14A cells (a neural progenitor cell line derived from the striatum of embryonic day 14 rats) endogenously expressing the other members of the ErbB family: ErbB1, ErbB2, and ErbB3. Using immunoprecipitation assays, we showed that the neuregulin-1beta1 (NRG1beta1) stimulus induced ErbB4 tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K) recruitment and activation (as demonstrated by Akt phosphorylation) either directly (ErbB4 cyt1 isoform) or indirectly (ErbB4 cyt2 isoform). We examined the ability of the four ErbB4 isoforms to induce chemotaxis and cell proliferation in response to NRG1beta1 stimulation. Using migration assays, we observed that only ErbB4-expressing cells stimulated with NRG1beta1 showed a significant increase in migration, whereas the growth rate remained unchanged. Additional assays showed that inhibition of PI3K (but not of phospholipase Cgamma) dramatically reduced migratory activity. Our data show that ErbB4 signaling via PI3K activation plays a fundamental role in controlling NRG1beta1-induced migration.

In the human fetus IGFBP-3 mRNA expression is most abundant in the skin, muscle and heart but circulating IGFBP-3 levels show age-related variations. In human heart tissues from controls and patients with either ischemic, dilated or hypertrophic cardiomyopathy (no.: 20, age-range from fetuses to elderly subjects) we determined the expression of cardiac IGFBP-3 mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) and the protein by Western blotting. The same parameters were also determined in human livers. We detected IGFBP-3 mRNA in neonatal and adult as well as in fetal human heart tissues in both ventricles. Western blotting revealed the presence of IGFBP-3 in all the examined cardiac tissues. IGFBP-3 appeared to be more abundant in the heart than in the liver and in the failing hearts from patients with ischemic heart disease than in those with hypertrophic cardiomyopathy. Thus both normal and pathological human heart tissues express IGFBP-3 across lifespan and IGFBP-3 could play IGF-dependent and/or -independent actions at the myocardial level.