The inclusion complex of 4-hydroxynonenal with a polymeric derivative of β-cyclodextrin enhances the antitumoral efficacy of the aldehyde in several tumor cell lines and in a three-dimensional human melanoma model

4-Hydroxynonenal (HNE) is the most studied end product of the lipoperoxidation process, by virtue of its relevant biological activity. The antiproliferative and proapoptotic effects of HNE have been widely demonstrated in a great variety of tumor cell types in vitro. Thus, it might represent a promising new molecule in anticancer therapy strategies. However, the extreme reactivity of this aldehyde, as well as its insolubility in water, a limiting factor for drug bioavailability, and its rapid degradation by specific enzymes represent major obstacles to its possible in vivo application. Various strategies can used to overcome these problems. One of the most attractive strategies is the use of nanovehicles, because loading drugs into nanosized structures enhances their stability and solubility, thus improving their bioavailability and their antitumoral effectiveness. Several natural or synthetic polymers have been used to synthesize nanosized structures and, among them, β-cyclodextrin (βCD) polymers are playing a very important role in drug formulation by virtue of the ability of βCD to form inclusion compounds with a wide range of solid and liquid molecules by molecular complexation. Moreover, several βCD derivatives have been designed to improve their physicochemical properties and inclusion capacities. Here we report that the inclusion complex of HNE with a derivative of βCD, the βCD–poly(4-acryloylmorpholine) conjugate (PACM-βCD), enhances the aldehyde stability. Moreover, the inclusion of HNE in PACM-βCD potentiates its antitumor effects in several tumor cell lines and in a more complex system, such as a human reconstructed skin carrying melanoma tumor cells.     

Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal

4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it.     

4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway

4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G₂/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested.     

4-Hydroxynonenal in the pathomechanisms of oxidative stress

Here we review the current knowledge on the biochemistry and molecular pathology of oxidative stress with specific regard to a major aldehydic end-product stemming from peroxidation of biomembranes, that is 4-hydroxynonenal (HNE). This multifunctional molecule, which derives from the most represented class of polyunsaturated fatty acids in the membranes, is potentially able to undergo a number of reactions with proteins, phospholipids, and nucleic acids. Despite an active metabolism in most of the cell types, HNE can be detected in several biological tissues by means of sufficiently precise methods, although with different sensitivity. In particular, relatively high steady-state levels of HNE are often detectable in a large variety of human disease processes, pointing to some involvement of the aldehyde in their pathogenesis. Among the prominent pathobiochemical effects of HNE is its remarkable stimulation of fibrogenesis and inflammation, which indicates a potential contribution of the aldehyde to the pathogenesis of several chronic diseases, whose progression is indeed supported by inflammatory reactions and characterized by fibrosis. Further, of interest appears to be the ability of HNE to modulate cell proliferation through interference with the activity of cyclins and protein kinases and with the apoptotic machinery. Finally, on the basis of the already achieved evidence, pursuing investigation of the role of HNE in signal transduction and gene expression seems very promising.     

4-Hydroxynonenal induces apoptosis in human osteoarthritic chondrocytes: the protective role of glutathione-S-transferase

Introduction

4-Hydroxynonenal (HNE) is one of the most abundant and reactive aldehydes of lipid peroxidation products and exerts various effects on intracellular and extracellular signalling cascades. We have previously shown that HNE at low concentrations could be considered as an important mediator of catabolic and inflammatory processes in osteoarthritis (OA). In the present study, we focused on characterizing the signalling cascade induced by high HNE concentration involved in cell death in human OA chondrocytes.

Methods

Markers of apoptosis were quantified with commercial kits. Protein levels were evaluated by Western blotting. Glutathione (GSH) and ATP levels were measured with commercial kits. Glucose uptake was assessed by 2-deoxy-D-[³H]-glucose. The role of GSH-S-transferase A4-4 (GSTA4-4) in controlling HNE-induced chondrocyte apoptosis was investigated by chondrocyte transfection with small interfering RNA (siRNA) or with the expression vector of GSTA4-4.

Results

Our data showed that HNE at concentrations of up to 10 μM did not alter cell viability but was cytotoxic at concentrations of greater than or equal to 20 μM. HNE-induced chondrocyte death exhibited several classical hallmarks of apoptosis, including caspase activation, cytochrome c and apoptosis-induced factor release from mitochondria, poly (ADP-ribose) polymerase cleavage, Bcl-2 downregulation, Bax upregulation, and DNA fragmentation. Our study of signalling pathways revealed that HNE suppressed pro-survival Akt kinase activity but, in contrast, induced Fas/CD95 and p53 expression in chondrocytes. All of these effects were inhibited by an antioxidant, N-acetyl-cysteine. Analysis of cellular energy and redox status showed that HNE induced ATP, NADPH, and GSH depletion and inhibited glucose uptake and citric acid cycle activity. GSTA4-4 ablation by the siRNA method augmented HNE cytotoxicity, but, conversely, its overexpression efficiently protected chondrocytes from HNE-induced cell death.

Conclusion

Our study provides novel insights into the potential mechanisms of cell death in OA cartilage and suggests the potential role of HNE in OA pathophysiology. GSTA4-4 expression is critically important for cellular defence against oxidative stress-induced cell death in OA cartilage, possibly by HNE elimination.     

Metabolic engineering for the optimization of hydrogen production in Escherichia coli: A review

Hydrogen is a potential sustainable energy source and it could become an alternative to fossil fuel combustion, thus helping to reduce greenhouse gas emissions. The biological production of hydrogen, instead of its chemical synthesis, is a promising possibility since this process requires less energy and is more sustainable and eco-friendly. Several microorganisms have been used for this purpose, but Escherichia coli is one of the most widely used in this field. The literature in this area has increased exponentially in the last 10 years and several strategies have been reported in an effort to improve hydrogen production. In this work, the stay of the art of hydrogen biosynthesis by E. coli and metabolic engineering strategies to enhance hydrogen production are reviewed. This work includes a discussion about the hydrogenase complexes responsible for the hydrogen synthesis in this microorganism and the central carbon metabolism pathways connected to this process. The main metabolic engineering strategies applied are discussed, including heterologous gene expression, adaptive evolution and metabolic and protein engineering. On the other hand, culture conditions, including the use of carbon sources such as glycerol, glucose or organic wastes, have also been considered. Yields and productivities of the most relevant engineered strains reported using several carbon sources are also compared.     

Regulation of 4-hydroxynonenal-mediated signaling by glutathione S-transferases

4-Hydroxynonenal (HNE), one of the major end products of lipid peroxidation, has been shown to be involved in signal transduction and available evidence suggests that it can affect cell cycle events in a concentration-dependent manner. Glutathione S-transferases (GSTs) can modulate the intracellular concentrations of HNE by affecting its generation during lipid peroxidation by reducing hydroperoxides and also by converting it into a glutathione conjugate. We have recently demonstrated that overexpression of the Alpha class GSTs in cells leads to lower steady-state levels of HNE, and these cells acquire resistance to apoptosis induced by lipid peroxidation-causing agents such as H(2)O(2), UVA, superoxide anion, and pro-oxidant xenobiotics, suggesting that signaling for apoptosis by these agents is transduced through HNE. Cells with the capacity to exclude HNE from the intracellular environment at a faster rate are relatively more resistant to apoptosis caused by H(2)O(2), UVA, superoxide anion, and pro-oxidant xenobiotics as well as by HNE, suggesting that HNE may be a common denominator in mechanisms of apoptosis caused by oxidative stress. We have also shown that transfection of adherent cells with HNE-metabolizing GSTs leads to transformation of these cells due to depletion of HNE. These recent studies from our laboratories, which strongly suggest that HNE is a key signaling molecule and that GSTs, being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article.     

Post-translational Modification of Serine/Threonine Kinase LKB1 via Adduction of the Reactive Lipid Species 4-Hydroxy-trans-2-nonenal (HNE) at Lysine Residue 97 Directly Inhibits Kinase Activity

Oxidative stress is pathogenic in a variety of diseases, but the mechanism by which cellular signaling is affected by oxidative species has yet to be fully characterized. Lipid peroxidation, a secondary process that occurs during instances of free radical production, may play an important role in modulating cellular signaling under conditions of oxidative stress. 4-Hydroxy-trans-2-nonenal (HNE) is an electrophilic aldehyde produced during lipid peroxidation that forms covalent adducts on proteins, altering their activity and function. One such target, LKB1, has been reported to be inhibited by HNE adduction. We tested the hypothesis that HNE inhibits LKB1 activity through adduct formation on a specific reactive residue of the protein. To elucidate the mechanism of the inhibitory effect, HEK293T cells expressing LKB1 were treated with HNE (10 μm for 1 h) and assayed for HNE-LKB1 adduct formation and changes in LKB1 kinase activity. HNE treatment resulted in the formation of HNE-LKB1 adducts and decreased LKB1 kinase activity by 31 ± 9% (S.E.) but had no effect on the association of LKB1 with its adaptor proteins sterile-20-related adaptor and mouse protein 25. Mutation of LKB1 lysine residue 97 reduced HNE adduct formation and attenuated the effect of HNE on LKB1 activity. Taken together, our results suggest that adduction of LKB1 Lys-97 mediates the inhibitory effect of HNE.     

4-hydroxynonenal as a bioactive marker of pathophysiological processes

The review is focused on the currently major aspect of 4-hydroxynonenal (HNE) research--studies that combine biological activities of the aldehyde together with the methods of its identification in cells and tissues. Because there were some excellent reviews on HNE published in recent years, starting in 1990 and 1991 with supreme reviews done by Hermann Esterbauer, who discovered the aldehyde, and colleagues from the Institute of Biochemistry in Graz, this article pays most of attention to the most recent articles, published in the last 15 months. Additionally, an overview on the relevance of HNE is given with respect to the research and publication trends in the period of 10 years (1993-2002) according to the data in the Current Contents and Medline data bases. It is obvious that HNE started in 1993 as a "toxic product of lipid peroxidation" and "second toxic messenger of free radicals", to become in 2002 a reliable marker of oxidative stress, a possible causative agent of several diseases (such as Alzheimer's disease), growth modulating factor and a signaling molecule. Novel analytical methods developed suitable pathways for HNE to become a clinically applicable marker of lipid peroxidation on one side and on the other a standardized parameter of food quality control. As it is also present physiologically in various cells and tissues, it is likely that HNE will soon become one of the most attractive factors for those who search for a small and reactive molecular link between genomics and proteomics.     

MicroRNA expression changes during human leukemic HL-60 cell differentiation induced by 4-hydroxynonenal,a product of lipid peroxidation

4-Hydroxynonenal (HNE) is one of several lipid oxidation products that may have an impact on human pathophysiology. It is an important second messenger involved in the regulation of various cellular processes and exhibits antiproliferative and differentiative properties in various tumor cell lines. The mechanisms by which HNE affects cell growth and differentiation are only partially clarified. Because microRNAs (miRNAs) have the ability to regulate several cellular processes, we hypothesized that HNE, in addition to other mechanisms, could affect miRNA expression. Here, we present the results of a genome-wide miRNA expression profiling of HNE-treated HL-60 leukemic cells. Among 470 human miRNAs, 10 were found to be differentially expressed between control and HNE-treated cells (at p < 0.05). Six miRNAs were down-regulated (miR-181a*, miR-199b, miR-202, miR-378, miR-454-3p, miR-575) and 4 were up-regulated (miR-125a, miR-339, miR-663, miR-660). Three of these regulated miRNAs (miR-202, miR-339, miR-378) were further assayed and validated by quantitative real-time RT-PCR. Moreover, consistent with the down-regulation of miR-378, HNE also induced the expression of the SUFU protein, a tumor suppressor recently identified as a target of miR-378. The finding that HNE could regulate the expression of miRNAs and their targets opens new perspectives on the understanding of HNE-controlled pathways. A functional analysis of 191 putative gene targets of miRNAs modulated by HNE is discussed.     

4-Hydroxy-2(E)-nonenal (HNE) catabolism and formation of HNE adducts are modulated by β oxidation of fatty acids in the isolated rat heart

We previously reported that a novel metabolic pathway functionally catabolizes 4-hydroxy-2(E)-nonenal (HNE) via two parallel pathways, which rely heavily on β-oxidation pathways. The hypothesis driving this report is that perturbations of β oxidation will alter the catabolic disposal of HNE, favoring an increase in the concentrations of HNE and HNE-modified proteins that may further exacerbate pathology. This study employed Langendorff perfused hearts to investigate the impact of cardiac injury modeled by ischemia/reperfusion and, in a separate set of perfusions, the effects of elevated lipid (typically observed in obesity and type II diabetes) by perfusing with increased fatty acid concentrations (1 mM octanoate). During ischemia, HNE concentrations doubled and the glutathione–HNE adduct and 4-hydroxynonanoyl-CoA were increased by 7- and 10-fold, respectively. Under conditions of increased fatty acid, oxidation to 4-hydroxynonenoic acid was sustained; however, further catabolism through β oxidation was nearly abolished. The inhibition of HNE catabolism was not compensated for by other disposal pathways of HNE, rather an increase in HNE-modified proteins was observed. Taken together, this study presents a mechanistic rationale for the accumulation of HNE and HNE-modified proteins in pathological conditions that involve alterations to β oxidation, such as myocardial ischemia, obesity, and high-fat diet-induced diseases.     

X-ray crystallographic analysis of adipocyte fatty acid binding protein (aP2) modified with 4-hydroxy-2-nonenal

Fatty acid binding proteins (FABP) have been characterized as facilitating the intracellular solubilization and transport of long‐chain fatty acyl carboxylates via noncovalent interactions. More recent work has shown that the adipocyte FABP is also covalently modified in vivo on Cys117 with 4‐hydroxy‐2‐nonenal (4‐HNE), a bioactive aldehyde linked to oxidative stress and inflammation. To evaluate 4‐HNE binding and modification, the crystal structures of adipocyte FABP covalently and noncovalently bound to 4‐HNE have been solved to 1.9 Å and 2.3 Å resolution, respectively. While the 4‐HNE in the noncovalently modified protein is coordinated similarly to a carboxylate of a fatty acid, the covalent form show a novel coordination through a water molecule at the polar end of the lipid. Other defining features between the two structures with 4‐HNE and previously solved structures of the protein include a peptide flip between residues Ala36 and Lys37 and the rotation of the side chain of Phe57 into its closed conformation. Representing the first structure of an endogenous target protein covalently modified by 4‐HNE, these results define a new class of in vivo ligands for FABPs and extend their physiological substrates to include bioactive aldehydes.     

Inhibition of the pyridine nucleotide-linked mitochondrial Ca2+ release by 4-hydroxynonenal: the role of thiolate-disulfide conversion

Summary

Rat liver mitochondria contain a specific Ca²⁺ release pathway which operates when intramitochondrial NAD⁺ is hydrolyzed to ADPribose and nicotinamide. The molecular details of this pathway are incompletely understood. It has been reported that NAD⁺ hydrolysis and therefore Ca²⁺ release stimulated by t-butylhydroperoxide is prevented by 4-hydroxynonenal (HNE). The reason underlying inhibition by HNE, however, remained unclear. It has also been reported that NAD⁺ hydrolysis and Ca²⁺ release are stimulated when some vicinal thiols are cross-linked, as shown with phenylarsine oxide or gliotoxin (GT). We now show that HNE also prevents the GT-induced Ca²⁺ release, but only when given before GT. Conversely, GT stimulates Ca²⁺ release only when given before HNE. Inhibition of Ca²⁺ release by HNE is reduced by its preincubation with thiol compounds, the effectiveness of which increases with decreasing pK_a of their sulfhydryl group. Preincubation of HNE with glutathione at high, but not at low, pH similarly reduces inhibition of Ca²⁺ release by HNE. These findings provide evidence that HNE inhibition of Ca²⁺ release is due to a modification of mitochondrial thiolates in a way that their cross-linking is prevented, and give further insight into the regulation of Ca²⁺ release from intact mitochondria.     

Role of 4-hydroxynonenal and its metabolites in signaling

Available evidence from a multitude of studies on the effects of 4-hydroxynonenal (HNE) on cellular processes seem to converge on some common themes: (i) concentration-dependent opposing effects of HNE on key signaling components (e.g. protein kinase C, adenylate cyclase) predict that certain constitutive levels of HNE may be needed for normal cell functions - lowering of this constitutive HNE level in cells promotes proliferative machinery while an increase in this level promotes apoptotic signaling; (ii) HNE is a common denominator in stress-induced apoptosis caused by H(2)O(2), superoxide, UV, heat or oxidant chemicals such as doxorubicin; and (iii) HNE can modulate ligand-independent signaling by membrane receptors such as EGFR or Fas (CD95) and may act as a sensor of external stimuli for eliciting stress-response. Against a backdrop of various reported effects of HNE, in vitro and in vivo, we have critically evaluated the above mentioned hypotheses suggesting a key role of HNE in signaling.     

4-Hydroxy-2,3-nonenal as a signal for cell function and differentiation.

4-Hydroxy-2,3-nonenal (HNE) is a biologically active aldehydic end product of oxidative decomposition of omega-3 and omega-6 polyunsaturated fatty acids of membrane phospholipids, a process referred to as lipid peroxidation. HNE has been detected in several experimental and clinical conditions in which oxidative stress has been reported to occur and several authors have suggested that HNE and related 4-hydroxy-2,3-alkenals (HAKs) of different chain length may act not only as toxic and mutagenic mediators of oxidative stress-related injury but also as biological signals in normal and pathological conditions. In this paper we will review the literature supporting the concept that HNE and HAKs may act as signal molecules able to modulate biological events such as chemotaxis, signal transduction, gene expression, cell proliferation and cell differentiation.     

Catabolism of (2E)-4-Hydroxy-2-nonenal via ω- and ω-1-Oxidation Stimulated by Ketogenic Diet

Oxidative stress triggers the peroxidation of ω-6-polyunsaturated fatty acids to reactive lipid fragments, including (2E)-4-hydroxy-2-nonenal (HNE). We previously reported two parallel catabolic pathways of HNE. In this study, we report a novel metabolite that accumulates in rat liver perfused with HNE or 4-hydroxynonanoic acid (HNA), identified as 3-(5-oxotetrahydro-2-furanyl)propanoyl-CoA. In experiments using a combination of isotopic analysis and metabolomics studies, three catabolic pathways of HNE were delineated following HNE conversion to HNA. (i) HNA is ω-hydroxylated to 4,9-dihydroxynonanoic acid, which is subsequently oxidized to 4-hydroxynonanedioic acid. This is followed by the degradation of 4-hydroxynonanedioic acid via β-oxidation originating from C-9 of HNA breaking down to 4-hydroxynonanedioyl-CoA, 4-hydroxyheptanedioyl-CoA, or its lactone, 2-hydroxyglutaryl-CoA, and 2-ketoglutaric acid entering the citric acid cycle. (ii) ω-1-hydroxylation of HNA leads to 4,8-dihydroxynonanoic acid (4,8-DHNA), which is subsequently catabolized via two parallel pathways we previously reported. In catabolic pathway A, 4,8-DHNA is catabolized to 4-phospho-8-hydroxynonanoyl-CoA, 3,8-dihydroxynonanoyl-CoA, 6-hydroxyheptanoyl-CoA, 4-hydroxypentanoyl-CoA, propionyl-CoA, and acetyl-CoA. (iii) The catabolic pathway B of 4,8-DHNA leads to 2,6-dihydroxyheptanoyl-CoA, 5-hydroxyhexanoyl-CoA, 3-hydroxybutyryl-CoA, and acetyl-CoA. Both in vivo and in vitro experiments showed that HNE can be catabolically disposed via ω- and ω-1-oxidation in rat liver and kidney, with little activity in brain and heart. Dietary experiments showed that ω- and ω-1-hydroxylation of HNA in rat liver were dramatically up-regulated by a ketogenic diet, which lowered HNE basal level. HET0016 inhibition and mRNA expression level suggested that the cytochrome P450 4A are main enzymes responsible for the NADPH-dependent ω- and ω-1-hydroxylation of HNA/HNE.     

Effect of 4-hydroxynonenal,a lipid peroxidation product,on exocytosis in HL-60 cells

Our work analysed the effect of 4-hydroxynonenal (HNE), a chemotactic aldehydic end-product of lipid peroxidation, on exocytosis in HL-60 cells. We measured the release of beta-glucuronidase, an enzyme of azurophil granules, from the cells incubated at 37 degrees C for 10 min in the presence of HNE concentrations ranging between 10(-8) and 10(-5) M. The release of lactate dehydrogenase was assayed to test cell viability. HNE (1 microM) was able to induce a significant and strong stimulation of beta-glucuronidase secretion without leading to cytotoxic effects. The finding that HNE could increase the exocytotic secretion from HL-60 cells together with its known chemotactic property supports the hypothesis that this lipid peroxidation product may play an important role as a chemical mediator of inflammation; moreover it is noteworthy that micromolar concentrations of HNE have actually been found in exudates from acute and chronic inflammations.     

The specificities of small molecule inhibitors of the TGFß and BMP pathways

Small molecule inhibitors of type 1 receptor serine threonine kinases (ALKs1-7), the mediators of TGFß and BMP signals, have been employed extensively to assess their physiological roles in cells and organisms. While all of these inhibitors have been reported as “selective” inhibitors of specific ALKs, extensive specificity tests against a wide array of protein kinases have not been performed. In this study, we examine the specificities and potencies of the most frequently used small molecule inhibitors of the TGFß pathway (SB-431542, SB-505124, LY-364947 and A-83-01) and the BMP pathway (Dorsomorphin and LDN-193189) against a panel of up to 123 protein kinases covering a broad spectrum of the human kinome. We demonstrate that the inhibitors of the TGFß pathway are relatively more selective than the inhibitors of the BMP pathway. Based on our specificity and potency profile and published data, we recommend SB-505124 as the most suitable molecule for use as an inhibitor of ALKs 4, 5 and 7 and the TGFß pathway. We do not recommend Dorsomorphin, also called Compound C, for use as an inhibitor of the BMP pathway. Although LDN-193189, a Dorsomorphin derivative, is a very potent inhibitor of ALK2/3 and the BMP-pathway, we found that it potently inhibited a number of other protein kinases at concentrations sufficient to inhibit ALK2/3 and its use as a selective BMP-pathway inhibitor has to be considered cautiously. Our observations have highlighted the need for caution when using these small molecule inhibitors to assess the physiological roles of BMP and TGFß pathways.