Thyroid Immune-Related Adverse Events in Patients with Cancer Treated with anti-PD1/anti-CTLA4 Immune Checkpoint Inhibitor Combination: Clinical Course and Outcomes

ObjectiveThyroid immune-related adverse events (irAEs) have been reported to have prognostic significance among patients with cancer treated with anti-programmed cell death-1 (PD1) and anti-programmed death-ligand 1 monotherapies. We evaluated the clinical course and predictors of thyroid irAEs in relation to outcomes of patients with advanced cancer treated with combination anti-PD1/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4).MethodsWe conducted a regional study and identified patients with advanced cancer who received ≥1 cycle of combination anti-PD1/anti-CTLA4 between 2015 and 2019 in Hong Kong. Thyroid function tests (TFTs) were monitored every 3 weeks. Thyroid irAE was defined by ≥2 abnormal TFTs after initiation of combination anti-PD1/anti-CTLA4 in the absence of other causes.ResultsOne hundred and three patients were included (median age: 59 years; 71.8% men). About 45% had prior anti-PD1 exposure. Upon median follow-up of 6.8 months, 17 patients (16.5%) developed thyroid irAEs, where 6 initially presented with thyrotoxicosis (overt, n = 4; subclinical, n = 2) and 11 with hypothyroidism (overt, n = 2; subclinical, n = 9). Eventually, 10 patients (58.8%) required continuous thyroxine replacement. Systemic steroid was not required in all cases. Prior anti-PD1 exposure (odds ratio, 3.67; 95% CI, 1.19–11.4; P = .024) independently predicted thyroid irAEs. Multivariable Cox regression analysis revealed that occurrence of thyroid irAEs was independently associated with better overall survival (adjusted hazard ratio, 0.34; 95% CI, 0.17–0.71; P = .004).ConclusionThyroid irAEs are common in routine clinical practice among patients with advanced cancer treated with anti-PD1/anti-CTLA4 combination and might have potential prognostic significance. Regular TFT monitoring is advised for timely treatment of thyroid irAEs to prevent potential morbidities.     

Vascular lipid droplets formed in response to TNF,hypoxia, or OA: biochemical composition and prostacyclin generation

Biogenesis of lipid droplets (LDs) in various cells plays an important role in various physiological and pathological processes. However, the function of LDs in endothelial physiology and pathology is not well understood. In the present work, we investigated the formation of LDs and prostacyclin (PGI₂) generation in the vascular tissue of isolated murine aortas following activation by proinflammatory factors: tumor necrosis factor (TNF), lipopolysaccharides (LPS), angiotensin II (AngII), hypoxic conditions, or oleic acid (OA). The abundance, size, and biochemical composition of LDs were characterized based on Raman spectroscopy and fluorescence imaging. We found that blockade of lipolysis by the adipose triglyceride lipase (ATGL) delayed LDs degradation and simultaneously blunted PGI₂ generation in aorta treated with all tested proinflammatory stimuli. Furthermore, the analysis of Raman spectra of LDs in the isolated vessels stimulated by TNF, LPS, AngII, or hypoxia uncovered that these LDs were all rich in highly unsaturated lipids and had a negligible content of phospholipids and cholesterols. Additionally, by comparing the Raman signature of endothelial LDs under hypoxic or OA-overload conditions in the presence or absence of ATGL inhibitor, atglistatin (Atgl), we show that Atgl does not affect the biochemical composition of LDs. Altogether, independent of whether LDs were induced by pro-inflammatory stimuli, hypoxia, or OA and of whether they were composed of highly unsaturated or less unsaturated lipids, we observed LDs formation invariably associated with ATGL-dependent PGI₂ generation. In conclusion, vascular LDs formation and ATGL-dependent PGI₂ generation represent a universal response to vascular proinflammatory insult.     

Targeted lipidomics reveals a novel role for glucosylceramides in glucose response

The addition of excess glucose to the diet drives a coordinated response of lipid metabolism pathways to tune the membrane composition to the altered diet. Here, we have employed targeted lipidomic approaches to quantify the specific changes in the phospholipid and sphingolipid populations that occur in elevated glucose conditions. The lipids within wild-type Caenorhabditis elegans are strikingly stable with no significant changes identified in our global mass spectrometry–based analysis. Previous work has identified ELO-5, an elongase that is critical for the synthesis of monomethyl branched-chain fatty acids (mmBCFAs), as essential for surviving elevated glucose conditions. Therefore, we performed targeted lipidomics on elo-5 RNAi-fed animals and identified several significant changes in these animals in lipid species that contain mmBCFAs as well as in species that do not contain mmBCFAs. Of particular note, we identified a specific glucosylceramide (GlcCer 17:1;O2/22:0;O) that is also significantly upregulated with glucose in wild-type animals. Furthermore, compromising the production of the glucosylceramide pool with elo-3 or cgt-3 RNAi leads to premature death in glucose-fed animals. Taken together, our lipid analysis has expanded the mechanistic understanding of metabolic rewiring with glucose feeding and has identified a new role for the GlcCer 17:1;O2/22:0;O.     

Importance of methodology in the evaluation of renal mononuclear phagocytes and analysis of a model of experimental nephritis with Shp1 conditional knockout mice

Tissue resident mononuclear phagocytes (Mophs), comprising monocytes, macrophages, and dendritic cells (DCs), play important roles under physiological and pathological conditions. The presence of these cells in the kidney has been known for decades, and studies of renal Mophs (rMophs) are currently underway. Since no unified procedure has been identified to isolate rMophs, results of flow cytometric analysis of rMophs have been inconsistent among studies. We therefore first evaluated a preparative method for rMophs using collagenous digestion. The yield of rMophs greatly increased after the collagenase digestion. In particular, F4/80^high rMophs, which were positive for CD11c, a specific marker of DCs, dramatically increased. In addition, since neutrophils are sometimes mixed among rMophs in the analysis of flow cytometry, we established a gating strategy for eliminating neutrophils. To determine the contribution of rMophs to the development of autoimmune nephritis, we analyzed an experimental model of autoimmune nephritis that was applied to Shp1 conditional knockout mice (Shp1 CKO). This knockout strain is generated by crossing a mouse line carrying floxed Shp1 allele to mice expressing Cre recombinase under the control of the CD11c promoter. Shp1 CKO therefore specifically lack Shp1 in cells expressing CD11c. As a result, Shp1 CKO were susceptible to that experimental glomerulonephritis and F4/80^high rMophs of Shp1 CKO increased dramatically. In conclusion, our preparative methods for collagenase digestion and gating strategy for neutrophils are necessary for the analysis of rMophs, and Shp1 suppresses the development of autoimmune nephritis through the control of rMophs.     

Pulmonary surfactant protein B carried by HDL predicts incident CVD in patients with type 1 diabetes

Atherosclerotic CVD is the major cause of death in patients with type 1 diabetes mellitus (T1DM). Alterations in the HDL proteome have been shown to associate with prevalent CVD in T1DM. We therefore sought to determine which proteins carried by HDL might predict incident CVD in patients with T1DM. Using targeted MS/MS, we quantified 50 proteins in HDL from 181 T1DM subjects enrolled in the prospective Coronary Artery Calcification in Type 1 Diabetes study. We used Cox proportional regression analysis and a case-cohort design to test associations of HDL proteins with incident CVD (myocardial infarction, coronary artery bypass grafting, angioplasty, or death from coronary heart disease). We found that only one HDL protein—SFTPB (pulmonary surfactant protein B)—predicted incident CVD in all the models tested. In a fully adjusted model that controlled for lipids and other risk factors, the hazard ratio was 2.17 per SD increase of SFTPB (95% confidence interval, 1.12–4.21, P = 0.022). In addition, plasma fractionation demonstrated that SFTPB is nearly entirely bound to HDL. Although previous studies have shown that high plasma levels of SFTPB associate with prevalent atherosclerosis only in smokers, we found that SFTPB predicted incident CVD in T1DM independently of smoking status and a wide range of confounding factors, including HDL-C, LDL-C, and triglyceride levels. Because SFTPB is almost entirely bound to plasma HDL, our observations support the proposal that SFTPB carried by HDL is a marker—and perhaps mediator—of CVD risk in patients with T1DM.     

Comparison of autophagy inducibility in various tyrosine kinase inhibitors and their enhanced cytotoxicity via inhibition of autophagy in cancer cells in combined treatment with azithromycin

Tyrosine kinase inhibitors (TKIs) induce autophagy in many types of cancer cells. We previously reported that gefitinib (GEF) and imatinib (IMA) induce autophagy in epidermal growth factor receptor (EGFR) knock-out A549 and non-BCR-ABL-expressing leukemia cell lines, respectively. This evidence suggests that TKI-induced autophagy is independent of the original target molecules. The present study compared the autophagy-inducing abilities of various TKIs, regardless of their targets, by quantitative autophagy flux assay. We established stable clones expressing the GFP-LC3-mCherry-LC3ΔG plasmid in A549, PC-9, and CAL 27 cell lines and assessed autophagy inducibility by monitoring the fluorescent ratios of GFP-LC3 to mCherry-LC3ΔG using an IncuCyte live cell imaging system during exposure to TKIs viz; GEF, osimertinib (OSI), lapatinib (LAP), lenvatinib (LEN), sorafenib (SOR), IMA, dasatinib (DAS), and tivantinib (TIV). Among these TKIs, DAS, GEF, and SOR exhibited prominent autophagy induction in A549 and PC-9 cells. In CAL 27 cells, IMA, SOR, and LEN, but not GEF, TIV, or OSI, exhibited autophagy induction. In the presence of azithromycin (AZM), which showed an inhibitory effect on autophagy flux, TKIs with prominent autophagy inducibility exhibited enhanced cytotoxicity via non-apoptotic cell death relative to effects of TKI alone. Therefore, autophagy inducibility of TKIs differed in the context of cancer cells. However, once induced, they appeared to have cytoprotective functions. Thus, blocking TKI-induced autophagy with AZM may improve the therapeutic effect of TKIs in cancer cells.     

Therapeutic potential of kaempferol on Streptococcus pneumoniae infection

Co-infections with pathogens and secondary bacterial infections play significant roles during the pandemic coronavirus disease 2019 (COVID-19) pathogenetic process, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Notably, co-infections with Streptococcus pneumoniae (S. pneumoniae), as a major Gram-positive pathogen causing pneumonia or meningitis, severely threaten the diagnosis, therapy, and prognosis of COVID-19 worldwide. Accumulating evidences have emerged indicating that S. pneumoniae evolves multiple virulence factors, including pneumolysin (PLY) and sortase A (SrtA), which have been extensively explored as alternative anti-infection targets. In our study, natural flavonoid kaempferol was identified as a potential candidate drug for infection therapeutics via anti-virulence mechanisms. We found that kaempferol could interfere with the pore-forming activity of PLY by engaging with catalytic active sites and consequently inhibit PLY-mediated cytotoxicity. Additionally, exposed to kaempferol significantly reduced the SrtA peptidase activity by occupying the active sites of SrtA. Further, the biofilms formation and bacterial adhesion to the host cells could be significantly thwarted by kaempferol incubation. In vivo infection model by S. pneumoniae highlighted that kaempferol oral administration exhibited notable treatment benefits, as evidenced by decreased bacterial burden, suggesting that kaempferol has tremendous potential to attenuate S. pneumoniae pathogenicity. Scientifically, our study implies that kaempferol is a promising therapeutic option by targeting bacterial virulence factors.     

10,12-Conjugated linoleic acid supplementation improves HDL composition and function in mice

Obesity is associated with inflammation, insulin resistance, and type 2 diabetes, which are major risk factors for CVD. One dietary component of ruminant animal foods, 10,12-conjugated linoleic acid (10,12 CLA), has been shown to promote weight loss in humans. Previous work has shown that 10,12 CLA is atheroprotective in mice by a mechanism that may be distinct from its weight loss effects, but this exact mechanism is unclear. To investigate this, we evaluated HDL composition and function in obese LDL receptor (Ldlr^?/?) mice that were losing weight because of 10,12 CLA supplementation or caloric restriction (CR; weight-matched control group) and in an obese control group consuming a high-fat high-sucrose diet. We show that 10,12 CLA-HDL exerted a stronger anti-inflammatory effect than CR- or high-fat high-sucrose-HDL in cultured adipocytes. Furthermore, the 10,12 CLA-HDL particle (HDL-P) concentration was higher, attributed to more medium- and large-sized HDL-Ps. Passive cholesterol efflux capacity of 10,12 CLA-HDL was elevated, as was expression of HDL receptor scavenger receptor class B type 1 in the aortic arch. Murine macrophages treated with 10,12 CLA in vitro exhibited increased expression of cholesterol transporters Abca1 and Abcg1, suggesting increased cholesterol efflux potential of these cells. Finally, proteomics analysis revealed elevated Apoa1 content in 10,12 CLA-HDL-Ps, consistent with a higher particle concentration, and particles were also enriched with alpha-1-antitrypsin, an emerging anti-inflammatory and antiatherosclerotic HDL-associated protein. We conclude that 10,12 CLA may therefore exert its atheroprotective effects by increasing HDL-P concentration, HDL anti-inflammatory potential, and promoting beneficial effects on cholesterol efflux.     

A systematic review of the natural history and biomarkers of primary lecithin:cholesterol acyltransferase deficiency

Syndromes associated with LCAT deficiency, a rare autosomal recessive condition, include fish-eye disease (FED) and familial LCAT deficiency (FLD). FLD is more severe and characterized by early and progressive chronic kidney disease (CKD). No treatment is currently available for FLD, but novel therapeutics are under development. Furthermore, although biomarkers of LCAT deficiency have been identified, their suitability to monitor disease progression and therapeutic efficacy is unclear, as little data exist on the rate of progression of renal disease. Here, we systematically review observational studies of FLD, FED, and heterozygous subjects, which summarize available evidence on the natural history and biomarkers of LCAT deficiency, in order to guide the development of novel therapeutics. We identified 146 FLD and 53 FED patients from 219 publications, showing that both syndromes are characterized by early corneal opacity and markedly reduced HDL-C levels. Proteinuria/hematuria were the first signs of renal impairment in FLD, followed by rapid decline of renal function. Furthermore, LCAT activity toward endogenous substrates and the percentage of circulating esterified cholesterol (EC%) were the best discriminators between these two syndromes. In FLD, higher levels of total, non-HDL, and unesterified cholesterol were associated with severe CKD. We reveal a nonlinear association between LCAT activity and EC% levels, in which subnormal levels of LCAT activity were associated with normal EC%. This review provides the first step toward the identification of disease biomarkers to be used in clinical trials and suggests that restoring LCAT activity to subnormal levels may be sufficient to prevent renal disease progression.     

Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.     

A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.     

Pharmacokinetics of Sublingual Versus Oral Estradiol in Transgender Women

ObjectiveTo investigate the pharmacokinetics of 17β-estradiol (E2) administered orally versus those of 17β-E2 administered sublingually in transgender women.MethodsSingle doses of 17β-E2 were administered orally (1 mg) to 10 transgender women and then sublingually (1 mg) after a 1-week washout period. Blood samples were collected at baseline (0 hour) and at 1, 2, 3, 4, 6, and 8 hours after dosing. The samples were frozen and analyzed using liquid chromatography mass spectrometry (LC-MS/MS) and immunoassay.ResultsThe results demonstrated that sublingual E2 had a significantly higher peak serum E2 concentration of 144 pg/mL, measured using LC-MS/MS, compared with an oral E2 concentration of 35 pg/mL, measured using LC-MS/MS (P = .003). Sublingual E2 peaked at 1 hour and oral E2 peaked at 8 hours, as measured using LC-MS/MS. The area under the curve (AUC) (0-8 hours) for sublingual E2, measured using LC-MS/MS, was 1.8-fold higher than the AUC (0-8 hours) for oral E2, measured using LC-MS/MS. Additionally, sublingual E2 was found to have an increased E2-to-estrone ratio at all time points (1.1 ± 1.0 vs 0.7 ± 0.4, P ≤ .0001), the clinical significance of which is unclear.ConclusionOral E2 administered sublingually has a different pharmacokinetic profile, with higher serum E2 levels and AUC (0-8 hours) than traditionally administered oral E2. Multidaily dosing may be necessary to suppress testosterone levels with sublingual E2. The appropriate dosing, efficacy, and safety of sublingual E2, compared with those of other E2 preparations, are unknown.     

Structural Basis of the Allosteric Inhibition of Human ABCG2 by Nanobodies

ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies.     

Determination of tissue contributions to the circulating lipid pool in cold exposure via systematic assessment of lipid profiles

Plasma lipid levels are altered in chronic conditions such as type 2 diabetes and cardiovascular disease as well as during acute stresses such as fasting and cold exposure. Advances in MS-based lipidomics have uncovered a complex plasma lipidome of more than 500 lipids that serve functional roles, including as energy substrates and signaling molecules. This plasma lipid pool is maintained through regulation of tissue production, secretion, and uptake. A major challenge in understanding the lipidome complexity is establishing the tissues of origin and uptake for various plasma lipids, which is valuable for determining lipid functions. Using cold exposure as an acute stress, we performed global lipidomics on plasma and in nine tissues that may contribute to the circulating lipid pool. We found that numerous species of plasma acylcarnitines (ACars) and ceramides (Cers) were significantly altered upon cold exposure. Through computational assessment, we identified the liver and brown adipose tissue as major contributors and consumers of circulating ACars, in agreement with our previous work. We further identified the kidney and intestine as novel contributors to the circulating ACar pool and validated these findings with gene expression analysis. Regression analysis also identified that the brown adipose tissue and kidney are interactors with the plasma Cer pool. Taken together, these studies provide an adaptable computational tool to assess tissue contribution to the plasma lipid pool. Our findings have further implications in understanding the function of plasma ACars and Cers, which are elevated in metabolic diseases.     

Autoantibodies against red blood cell antigens are common in a malaria endemic area

Plasmodium falciparum malaria can cause severe anemia. Even after treatment, hematocrit can decrease. The role of autoantibodies against erythrocytes is not clearly elucidated and how common they are, or what they are directed against, is still largely unknown.We have investigated antibodies against erythrocytes in healthy adult men living in a highly malaria endemic area in Uganda. We found antibodies in more than half of the individuals, which is significantly more than in a non-endemic area (Sweden). Some of the Ugandan samples had a broad reactivity where it was not possible to determine the exact target of the autoantibodies, but we also found specific antibodies directed against erythrocyte surface antigens known to be of importance for merozoite invasion such as glycophorin A (anti-En^a, anti-M) and glycophorin B (anti-U, anti-S). In addition, several autoantibodies had partial specificities against glycophorin C and the blood group systems Rh, Diego (located on Band 3), Duffy (located on ACKR1), and Cromer (located on CD55), all of which have been described to be important for malaria and therefore of interest for understanding how autoantibodies could potentially stop parasites from entering the erythrocyte.In conclusion, specific autoantibodies against erythrocytes are common in a malaria endemic area.     

Red Blood Cells Protein Profile Is Modified in Breast Cancer Patients

Metastasis is the primary cause of death for most breast cancer (BC) patients who succumb to the disease. During the hematogenous dissemination, circulating tumor cells interact with different blood components. Thus, there are microenvironmental and systemic processes contributing to cancer regulation. We have recently published that red blood cells (RBCs) that accompany circulating tumor cells have prognostic value in metastatic BC patients. RBC alterations are related to several diseases. Although the principal known role is gas transport, it has been recently assigned additional functions as regulatory cells on circulation. Hence, to explore their potential contribution to tumor progression, we characterized the proteomic composition of RBCs from 53 BC patients from stages I to III and IV, compared with 33 cancer-free controls. In this work, we observed that RBCs from BC patients showed a different proteomic profile compared to cancer-free controls and between different tumor stages. The differential proteins were mainly related to extracellular components, proteasome, and metabolism. Embryonic hemoglobins, not expected in adults’ RBCs, were detected in BC patients. Besides, lysosome-associated membrane glycoprotein 2 emerge as a new RBCs marker with diagnostic and prognostic potential for metastatic BC patients. Seemingly, RBCs are acquiring modifications in their proteomic composition that probably represents the systemic cancer disease, conditioned by the tumor microenvironment.     

Glycosylation is a novel TGFβ1-independent post-translational modification of Smad2

Smad2 is a crucial component of intracellular signaling by transforming growth factor-β (TGFβ). Here we describe that Smad2 is glycosylated, which is a novel for Smad2 post-translational modification. We showed that the Smad2 glycosylation was inhibited upon treatment of cells with 17β-estradiol, and was enhanced in cells in a dense culture as compared to cells in a sparse culture. The Smad2 glycosylation was not dependent on the C-terminal phosphorylation of Smad2, and was not affected by TGFβ1 treatment of the cells. Smad2 was glycosylated at multiple sites, and one of the predicted sites is Serine110. Thus, Smad2 is glycosylated, and this post-translational modification was modulated by 17β-estradiol but not by TGFβ1.