Secreted Frizzled-related Protein 4 Research Articles

Finding Novel Anti-carcinomas Compounds by Targeting SFRP4 Through Molecular Modeling, Docking and Dynamic Simulation Studies.

Secreted Frizzled-Related Protein 4 (SFRP4) is a glycoprotein that acts as a competitor of both canonical and non-canonical Wnt pathways. SFRP4 is mostly expressed in ovary and plays a significant role as a target molecule to cure ovarian carcinoma. Multiple chemical agonists are being used to cure ovary melanoma. We are interested in theoretically analyzing the compounds through computational approaches for their potential inhibitory effects against SFRP4. Compounds were sketched in Chemsketch drawing tool and minimized through chimera tool. Because the crystal structure of SFRP4 is not available in Protein Data Bank, homology modeling approach was used to predict Three-Dimensional (3D) crystal structure of SFRP4. Moreover, multiple computational approaches such as molecular docking and Molecular Dynamic (MD) simulations along with various online tools were employed to screen the best inhibitor against ovary melanoma. The docking results showed that 1d and 1e compounds revealed significant binding energy values (-9.10 and -9.00 kcal/mol, respectively) compared with the standard drugs such as cis-platin and docetaxel (-3.30, -10.80 kcal/mol), respectively. Moreover, MD simulation results showed that 1d has little fluctuations throughout the simulation period as depicted by the root mean square deviation and root mean square fluctuation graphs. The present in-silico study provides a deeper insight into the structural attributes of 1d compound and its overall molecular interactions against SFRP4 and gives a hypothetical gateway to use this compound as a potential inhibitor against ovarian carcinoma.

Morphology-oriented epigenetic research.

Cytosine methylation plays a major role in the regulation of sequential and tissue-specific expression of genes. De novo aberrant DNA methylation and demethylation are also crucial processes in tumorigenesis and tumor progression. The mechanisms of how and when such aberrant methylation and demethylation occur in tumor cells are still obscure, however. To evaluate subtle epigenetic alteration among minor subclonal populations, morphology-oriented epigenetic analysis is requisite, especially where heterogeneity and flexibility are as notable as in the process of cancer progression and cellular differentiation at critical stages. Therefore, establishment of reliable morphology-oriented epigenetic studies has become increasingly important in not only the experimental but also the diagnostic field. By selecting a subset of cells based on characteristic morphological features disclosed by microdissection or in situ hybridization, we discovered how methylation at certain CpG sites outside of CpG islands would play a crucial epigenetic role in the versatility and flexibility of gene expression during cancer progression. In this review, we first introduce technical aspects of two morphology-oriented epigenetic studies: (1) histoendonuclease-linked detection of methylated sites of DNA (HELMET), and (2) padlock probe and rolling circle amplification (RCA) for in situ identification of methylated cytosine in a sequence-dependent manner. We then present our observation of a novel MeCP2-mediated gene-silencing mechanism through the addition of methylation to a single-CpG-locus upstream of the TATA-box of the receptor activator of NF-κB ligand (RANKL) and of secreted frizzled-related protein 4 (SFRP4) gene promoters.

Idiopathic infertility in women is associated with distinct changes in proliferative phase uterine fluid proteins†

The regenerative, proliferative phase of a woman's menstrual cycle is a critical period which lays the foundation for the subsequent, receptive secretory phase. Although endometrial glands and their secretions are essential for embryo implantation and survival, the proliferative phase, when these glands form, has been rarely examined. We hypothesized that alterations in the secreted proteome of the endometrium of idiopathic infertile women would reflect a disturbance in proliferative phase endometrial regeneration. Our aim was to compare the proteomic profile of proliferative phase uterine fluid from fertile (n=9) and idiopathic infertile (n=10) women. Proteins with≥2-fold change (P<0.05) were considered significantly altered between fertile and infertile groups. Immunohistochemistry examined the endometrial localization of identified proteins. Western immunoblotting defined the forms of extracellular matrix protein 1 (ECM1) in uterine lavage fluid. Proteomic analysis identified four proteins significantly downregulated in infertile women compared to fertile women, including secreted frizzled-related protein 4 (SFRP4), CD44, and ECM1: two proteins were upregulated. Seven proteins were unique to the fertile group and six (including isoaspartyl peptidase/L-asparaginase [ASRGL1]) were unique to the infertile group. Identified proteins were classified into biological processes of tissue regeneration and regulatory processes. ASRGL1, SFRP4, and ECM1 localized to glandular epithelium and stroma, cluster of differentiation 44 (CD44) to stroma and immune cells. ECM1 was present in two main molecular weight forms in uterine fluid. Our results indicate a disturbance in endometrial development during the proliferative phase among infertile women, providing insights into human endometrial development and potential therapeutic targets for infertility.

Differential Patterns of Secreted Frizzled-Related Protein 4 (SFRP4) in Adipocyte Differentiation: Adipose Depot Specificity

Background/Aims: Secreted frizzled-related protein 4 (SFRP4) is a member of the SFRP family that acts as soluble modulators of Wnt signaling. Given the substantial rise in obesity, depot-specific fat accumulation and its associated diseases like diabetes, it is important to understand the molecular basis of depot-specific adipocyte differentiation. In the current study, we investigated the expression of SFRP4 in both subcutaneous and visceral adipose tissue in terms of their differentiation. Methods: White preadipocytes were isolated from the inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) from C57BL/6J mice (age: 8-week-old, male). SFRP4 expression in iWAT and eWAT preadipocytes was silenced by siRNA transfection and harvested cells for gene and protein expression analysis was performed during the differentiation. Furthermore, iWAT and eWAT preadipocytes treated with or without IL-1β were harvested for gene and protein expression analysis. Results: SFRP4 expression levels were gradually increased and proportionally associated with eWAT adipocyte differentiation toward maturation at 14 days, while iWAT adipocyte just showed an opposite tendency. Moreover, genetic (adiponectin, C/EBPα, C/EBPβ, FABP4, GLUT4 and PPARγ) analysis demonstrated that depot-specific adipogenesis in response to SFRP4 silencing in eWAT and iWAT preadipocytes. Upon IL-1β treatment, SFRP4 mRNA expression decreased significantly in iWAT adipocyte, but the expression was no significant difference in eWAT adipocyte. Conclusion: These results suggest that SFRP4 expression differentially mediates adipocyte differentiation and may play an important role in adipogenesis.

Increased secreted frizzled-related protein 4 and ficolin-3 levels in gestational diabetes mellitus women.

By biochemical and epidemiological similarity with type 2 diabetes mellitus (T2DM), gestational diabetes mellitus (GDM) has some overlap between prediction markers and risk factors of T2DM. The present study aimed to establish that secreted frizzled-related protein 4 (SFRP4) and ficolin-3 levels, which have been linked to insulin resistance and the development of T2DM, are elevated in GDM women. A longitudinal prospective cohort study of 86 GDM and 273 normal glucose tolerant (NGT) pregnant women was performed. The clinical parameters, lipid profiles, and serum SFRP4 and ficolin-3 levels were tested during the early and late second-trimester and third-trimester of pregnancy. Both SFRP4 and ficolin-3 levels were significantly higher in GDM women as compared to the NGT participants at three test points (p < 0.01). Spearman's correlation analysis showed that serum SFRP4 levels were significantly positively correlated with ficolin-3 during the early and late second-trimester and third-trimester of pregnancy. The elevated SFRP4 and ficolin-3 concentrations at 16-18 weeks gestation significantly associated with GDM were conformed using binary logistic regression analysis after controlling for other variables [odds ratios (OR) with 95% confidence intervals (CI) for SFRP4: 2.84 (1.78-4.53), p < 0.01; for ficolin-3: 2.45 (1.55-3.88), p < 0.01]. In Conclusions, increased SFRP4 and ficolin-3 levels are significantly associated with GDM development and might be important risk factors for this pregnancy complication.

Prorenin and secreted frizzled-related protein 4 levels in women with gestational diabetes mellitus.

This study was designed to investigate prorenin and secreted frizzled-related protein 4 (SFRP4) levels in pregnancies with or without gestational diabetes mellitus (GDM). A total of 76 pregnant women were included in the study. Thirty-five of the pregnant women were included in GDM group according to the results of oral glucose tolerance tests (OGTT) and 41 of them were included in the control group. In the group with GDM, SFRP4 value was found to be significantly higher than that of the control group (5.59 ± 3.32 ng/mL vs 4.05 ± 2.15 ng/mL; p = 0.017). Women with GDM had significantly higher serum prorenin levels compared with control group [737 (427-1339) pg/mL vs. 535 (376-725) pg/mL; p = 0.009]. There was a significant positive association between prorenin and SFRP4 levels in GDM (r = 0.91; p < 0.001) and control groups (r = 0.42; p = 0.002) and whole pregnancies (r = 0.75; p = 0.002). We have shown that prorenin and SFRP4 were significantly elevated in GDM patients when compared to healthy control group. Furthermore, we found that there was a positive correlation between prorenin and SFRP4 (Tab. 1, Fig. 2, Ref. 38).

Evaluation of fractalkine (FKN) and secreted frizzled-related protein 4 (SFRP-4) serum levels in patients with prediabetes and type 2 diabetes.

The objective of this study is to compare serum levels of FKN and SFRP-4 in patients with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and type 2 diabetes mellitus (T2DM). A total of 152 patients presented to the endocrinology outpatient clinic of our hospital were included in the study. Eighty-two patients with a history of T2DM were assigned to the T2DM group. IGT (n = 34) and NGT (n = 36) groups included the patients who received oral glucose tolerance test outcomes. Serum FKN levels were significantly higher in the IGT and T2DM groups compared to the NGT group (p< 0.001 and p < 0.001, respectively). Serum SFRP-4 levels were significantly higher in the T2DM group compared to the IGT and NGT groups (p = 0.001 and p = 0.004, respectively). A significant correlation was observed between FKN and fasting glucose levels. SFRP-4 was significantly correlated with fasting glucose, HbA1c, and triglyceride levels. To our knowledge, increased FKN levels in patients with IGT were demonstrated for the first time in this study. The results of our study support the opinion that FKN and SFRP-4 may contribute to the pathogenesis of T2DM (Tab. 1, Fig. 3, Ref. 23).

Delivery of expression constructs of secreted frizzled-related protein 4 and its domains by chitosan-dextran sulfate nanoparticles enhances their expression and anti-cancer effects.

In malignant mesothelioma (MM) cells, secreted frizzled-related protein 4 (SFRP4) expression is downregulated by promoter methylation. In this study, we evaluated the effect of encapsulated chitosan-dextran (CS-DS) nanoparticle formulations of SFRP4 and its cysteine-rich domain (CRD) and netrin-like domain (NLD) as means of SFRP4-GFP protein delivery and their effects in JU77 and ONE58 MM cell lines. CS-DS formulations of SFRP4, CRD, and NLD nanoparticles were prepared by a complex coacervation technique, and particle size ranged from 300nm for empty particles to 337nm for particles containing the proteins. Measurement of the zeta potential showed that all preparations were around 25mV or above, suggesting stable formulation and good affinity for the DNA molecules. The CS-DS nanoparticle formulation maintained high integrity and entrapment efficiency. Gene delivery of SFRP4 and its domains showed enhanced biological effects in both JU77 and ONE58 cell lines when compared to the non-liposomal FUGENE® HD transfection reagent. In comparison to the CRD nanoparticles, both the SFRP4 and NLD nanoparticles significantly reduced the viability of MM cells, with the NLD showing the greatest effect. The CS-DS nanoparticle effects were observed at an earlier time point and with lower DNA concentrations. Morphological changes in MM cells were characterized by the formation of membrane-associated vesicles and green fluorescent protein expression specific to SFRP4 and the NLD. The findings from our proof-of-concept study provide a stepping stone for further investigations using in vivo models.

SFRP4 gene expression is increased in aggressive prostate cancer

Increased knowledge of the molecular differences between indolent and aggressive prostate cancer is needed for improved risk stratification and treatment selection. Secreted frizzled-related protein 4 (SFRP4) is a modulator of the cancer-associated Wnt pathway, and previously suggested as a potential marker for prostate cancer aggressiveness. In this study, we investigated and validated the association between SFRP4 gene expression and aggressiveness in nine independent cohorts (n = 2157). By differential expression and combined meta-analysis of all cohorts, we detected significantly higher SFRP4 expression in cancer compared with normal samples, and in high (3–5) compared with low (1–2) Grade Group samples. SFRP4 expression was a significant predictor of biochemical recurrence in six of seven cohorts and in the overall analysis, and was a significant predictor of metastatic event in one cohort. In our study cohort, where metabolic information was available, SFRP4 expression correlated significantly with the concentrations of citrate and spermine, two previously suggested biomarkers for aggressive prostate cancer. SFRP4 immunohistochemistry in an independent cohort (n = 33) was not associated with aggressiveness. To conclude, high SFRP4 gene expression is associated with high Grade Group and recurrent prostate cancer after surgery. Future studies investigating the mechanistic and clinical usefulness of SFRP4 in prostate cancer are warranted.

Glucagon-like peptide-1 levels and dipeptidyl peptidase-4 activity in type 2 diabetes.

Hyperglycemia is the major risk factor for microvascular complications in type 2 diabetes mellitus (T2DM) patients. This randomized controlled clinical trial aimed to investigate T2DM patients with microvascular complications with regard to possible relations among serum clusterin (CLU), amylin, secreted frizzled-related protein-4 (SFRP-4), glucagon-like peptide-1 (GLP-1) and dipeptidyl peptidase-4 (DPP-4) activities. Subject groups were defined as follows: T2DM without complications (n=25, F/M=9/16, age 53.9±11.1 years); T2DM+Retinopathy (n=25, F/M=13/12, age 63.8±7.1 years); T2DM+Nephropathy (n=25, F/M=13/12, age 58.7±14.4 years); T2DM+Neuropathy (n=25, F/M=15/10, age 63.2±9.6 years); and healthy control subjects (HC) (n=25). CLU, amylin, SFRP-4, DPP-4 and GLP-1 (total and active) activities were measured and compared in blood samples from type 2 diabetic patients with and without microvascular complications. Significantly lower levels of DPP-4 and GLP-1total (P.

Human epicardial adipose tissue-derived and circulating secreted frizzled-related protein 4 (SFRP4) levels are increased in patients with coronary artery disease

BackgroundPrevious studies have demonstrated that secreted frizzled-related protein 4 (SFRP4) is associated with impaired glucose and triglyceride metabolism in patients with stable coronary artery disease. In the present study, we investigated human epicardial adipose tissue (EAT)-derived and circulating SFRP4 levels in patients with coronary artery disease (CAD).MethodsPlasma samples and adipose biopsies from EAT and subcutaneous adipose tissue (SAT) were collected from patients with CAD (n = 40) and without CAD (non-CAD, n = 30) during elective cardiac surgery. The presence of CAD was identified by coronary angiography. SFRP4 mRNA and protein expression levels in adipose tissue were detected by quantitative real-time PCR and immunohistochemistry, respectively. Plasma SFRP4 concentrations were measured by an enzyme-linked immunosorbent assay (ELISA). Correlation analysis and multivariate linear regression analysis were used to determine the association of SFRP4 expression with atherosclerosis as well as clinical risk factors.ResultsSFRP4 mRNA and protein expression levels were significantly lower in EAT than in paired SAT in patients with and without CAD (all P < 0.05). Compared to non-CAD patients, CAD patients had higher SFRP4 expression levels in EAT (both mRNA and protein levels) and in plasma. Multivariate linear regression analysis showed that CAD was an independent predictor of SFRP4 expression levels in EAT (beta = 0.442, 95% CI 0.030–0.814; P = 0.036) and in plasma (beta = 0.300, 95% CI 0.056–0.545; P = 0.017). SAT-derived SFRP4 mRNA levels were independently associated with fasting insulin levels (beta = 0.382, 95% CI 0.008–0.756; P = 0.045). In addition, plasma SFRP4 levels were positively correlated with BMI (r = 0.259, P = 0.030), fasting insulin levels (r = 0.306, P = 0.010) and homeostasis model assessment of insulin resistance (HOMA-IR) values (r = 0.331, P = 0.005).ConclusionsEAT-derived and circulating SFRP4 expression levels were increased in patients with CAD. EAT SFRP4 mRNA levels and plasma SFRP4 concentrations were independently associated with the presence of CAD.

GW28-e0895 Association of secreted frizzled-related protein 4 (SFRP4) expression in human epicardial adipose tissue with coronary atherosclerosis

GW28-e0895 Association of secreted frizzled-related protein 4 (SFRP4) expression in human epicardial adipose tissue with coronary atherosclerosis

Overexpression of miR-135b-5p promotes unfavorable clinical characteristics and poor prognosis via the repression of SFRP4 in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and malignant neoplasm. The aberrant expression of miR-135b-5p and secreted frizzled-related protein 4 (SFRP4) has been revealed to be involved in various cancers. However, the clinical significance of miR-135b-5p and that of its potential target SFRP4 in PDAC remain to be elucidated. Here, we found that miR-135b-5p was markedly upregulated in pancreatic cancer tissue compared with corresponding adjacent normal tissue, whereas SFRP4 was significantly downregulated. The expression of miR-135b-5p was negatively correlated with the expression of SFRP4. PDAC patients with regional lymph node metastases, vascular invasion, tumor microthrombus and higher PET-CT SUVmax values had significantly higher expression of miR-135b-5p. Immunoblotting revealed that regional lymph node metastases were correlated with expressive states of SFRP4. Negative SFRP4 expression was significantly associated with old age, larger tumor size, regional lymph node metastasis and poor differentiation. Survival analyses demonstrated that miR-135b-5p and SFRP4 could predict outcomes and that miR-135b-5p was an independent predictor. In vitro, the overexpression of miR-135b-5p promoted the migration and proliferation of PANC-1 and MiaPaCa-2 cells, while immunoblotting demonstrated the downregulation of SFRP4 and the upregulation of beta-catenin. Inhibition of miR-135b-5p suppressed migration, induced apoptosis of PANC-1 and AsPC-1 cells, and reduced the expression of beta-catenin. A luciferase reporter assay confirmed that miR-135b-5p repressed the expression of SFRP4 via the direct targeting of its 3’-untranslated regions. In conclusion, the overexpression of miR-135b-5p and the downregulation of SFRP4 were associated with unfavorable clinical characteristics and poor prognosis, and SFRP4 was shown to be a direct downstream target of miR-135b-5p. Thus, the mechanism that underlies the miR-135b-5p-SFRP4-Wnt/beta-catenin axis represents a potential target for PDAC diagnosis and therapy.

Abstract 932: Wnt antagonist, secreted frizzled-related protein 4 (SFRP4) and peptides from its associated domains, increases chemotherapeutic response of cancer stem cells of malignant mesothelioma cell lines

Abstract Malignant mesothelioma (MM) is a highly aggressive cancer associated with past asbestos exposure that is characterized by rapid progression, late metastases and poor prognosis. There is a need for much improved and novel therapies for this cancer through improved biological understanding of the disease.Previous studies have shown that SFRPs in general and SFRP4 in particular are down regulated in mesothelioma tissue and cell lines 1-4. Based on our own study and previously demonstrated a role for sFRP4 in making chemoresistant tumours amenable to chemotherapeutics 5-16. We have now progressed our work into the effects of peptides derived from the cysteine-rich domain and the netrin-like domain of sFRP4 on tumour initiating cells derived from MM cell lines.We examined the effect of secreted frizzled-related protein 4 (sFRP4), a Wnt signaling antagonist and its associated peptides, in chemosensitizing the MM cell line ONE 58 and JU77 and MM cancer stem cells (CSCs) enriched from these two cell lines to chemotherapeutics.We found tumorspheres exposed to sFRP4 and peptides alone and in combination with cisplatin induced cell death and decreased CD133 and ALDH1 expression.We thus identified for the first time that sFRP4 and associated peptides could help to sensitise to chemotherapeutics and destroy cancer stem cells of MM cell line, which would lead to effective treatment regimen to combat malignant mesothelioma. Our initial in vitro data exemplify the ability of these peptides in diminishing the numbers of therapy-resistant Tumor initiating cells (TICs).

HCG activates Epac-Erk1/2 signaling regulating Progesterone Receptor expression and function in human endometrial stromal cells.

How does hCG signal in human endometrial stromal cells (ESCs) and what is its role in regulating ESC function? hCG signaling in ESCs activates the extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) pathway through exchange protein activated by cyclic AMP (cAMP) (Epac) and transiently increases progesterone receptor (PR) transcript and protein expression and its transcriptional function. hCG is one of the earliest embryo-derived secreted signals in the endometrium, which abundantly expresses LH/hCG receptors. hCG signals through cAMP/protein kinase A (PKA) in gonadal cells, but in endometrial epithelial cells, hCG induces Erk1/2 activation independent of the cAMP/PKA pathway. Few data exist concerning the signal transduction pathways triggered by hCG in ESCs and their role in regulation of ESC function. This is an in vitro study comprising patients undergoing benign gynecological surgery (n = 46). Endometrial samples were collected from normal cycling women during the mid-secretory phase for ESCs isolation. The study conducted in an academic research laboratory within a tertiary-care hospital. The activation of the Erk1/2 signal transduction pathway elicited by hCG was evaluated in ESC. Signaling pathway inhibitors were used to examine the roles of PKA, PI3K, PKC, adenylyl cyclase and Epac on the hCG-stimulated up-regulation of phospho-Erk1/2 (pErk1/2). Erk1/2 phosphorylation was determined by immunoblot. siRNA targeting Epac was used to investigate the molecular mechanisms. To assess the role of Erk1/2 signaling induced by hCG on ESC function, gene expression regulation was examined by immunofluorescence and real-time quantitative PCR. The role of PR on the regulation of transcript levels was studied using progesterone and the PR antagonist RU486. All experiments were conducted using at least three different cell culture preparations in triplicate. Addition of hCG to ESCs in vitro induced the phosphorylation of Erk1/2 through cAMP accumulation. Such induction could not be blocked by inhibitors for PKA, PKC and PI3K. Epac inhibition and knockdown with siRNA prevented pErk1/2 induction by hCG. ESCs stimulated with hCG for up to 72 h showed a significant increase in PR mRNA and immunofluorescent label at 48 h only; an effect that was abrogated with the mitogen-activated protein kinase kinase inhibitor UO126. In addition, the hCG-activated Erk1/2 pathway significantly decreased the mRNA levels for secreted frizzled-related protein 4 (SFRP4) at 24 h, whereas it increased those for homeobox A10 (HOXA10) at 48 h (P = 0.041 and P = 0.022 versus control, respectively). Prolactin mRNA levels were not significantly modified. HOXA10 mRNA up-regulation by hCG was not enhanced by co-stimulation with progesterone; however, it was completely abolished in the presence of RU486 (P = 0.036 hCG versus hCG + RU486). N/A. This is an in vitro study utilizing stromal cell cultures from human endometrial tissues. Furthermore, results obtained should also be confirmed in vivo in the context of the whole human endometrial tissue and hormonal milieu. The in vitro experiments using hCG have been conducted without other hormones/factors that may also modulate the ESCs response to hCG. We have determined that hCG induces the PR through the Erk1/2 pathway in ESCs which may render them more sensitive to progesterone, increasing our understanding about the effects of hCG at the embryo-maternal interface. The activation of such a pathway in the context of the hormonal milieu during the window of implantation might contribute to a successful dialog between the embryo and the uterus, leading to appropriate endometrial function. Defective hCG signaling in the endometrial stromal tissue may lead to an incomplete uterine response, compromising embryo implantation and early pregnancy. This work was supported by the National Fund for Scientific and Technological Development, Government of Chile (FONDECYT) grants 11100443 and 1140614 (A.T.-P.). The authors have no conflicts of interest to declare.

Sequencing and bioinformatics analysis of the differentially expressed genes in herniated discs with or without calcification.

The purpose of this study was to detect the differentially expressed genes between ossified herniated discs and herniated discs without ossification. In addition, we sought to identify a few candidate genes and pathways by using bioinformatics analysis. We analyzed 6 samples each of ossified herniated discs (experimental group) and herniated discs without ossification (control group). Purified mRNA and cDNA extracted from the samples were subjected to sequencing. The NOISeq method was used to statistically identify the differentially expressed genes (DEGs) between the 2 groups. An in-depth analysis using bioinformatics tools based on the DEGs was performed using Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein-protein interaction network analysis. The top 6 DEGs were verified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A total of 132 DEGs was detected. A total of 129 genes in the ossified group were upregulated and 3 genes were found to be downregulated as compared to the control group. The top 3 cellular components in GO ontologies analysis were extracellular matrix components. GO functions were mainly related to the glycoprotein in the cell membrane and extracellular matrix. The GO process was related to completing response to stimulus, immune reflex and defense. The top 5 KEGG enrichment pathways were associated with infection and inflammation. Three of the top 20 DEGs [sclerostin (SOST), WNT inhibitory factor 1 (WIF1) and secreted frizzled related protein 4 (SFRP4)] were related to the inhibition of the Wnt pathway. The ossified discs exhibited a higher expression of the top 6 DEGs [SOST, joining chain of multimeric IgA and IgM (IGJ; also known as JCHAIN), defensin alpha 4 (DEFA4), SFRP4, proteinase 3 (PRTN3) and cathepsin G (CTSG)], with the associated P-values of 0.045, 0.000, 0.008, 0.010, 0.015 and 0.002, respectively, as calculated by the independent sample t-test. The gene expression profiling of the 2 groups revealed differential gene expression. Thus, our data suggest that Wnt pathway abnormality and local inflammation may be related to disc ossification.

Cross-talk of SFRP4, integrin α1β1, and Notch1 inhibits cardiac differentiation of P19CL6 cells

Signaling pathways play an important role in cardiogenesis. Secreted frizzled-related protein 4 (SFRP4), a member of the Wnt family, contributes to adipogenesis and tumorigenesis. However, how SFRP4 participates in cardiogenesis and the detailed molecular mechanisms involved have not been elucidated. The aim of this work was to determine cross-talk between SFRP4, integrin α1β1, and Notch1 during cardiac differentiation of P19CL6 cells. Using a well-established in vitro P19CL6 cell cardiomyocyte differentiation system, we found that SFRP4 inhibited P19CL6 cell cardiac differentiation via SFRP4 overexpression or knockdown. In addition, the SFRP4 overexpression augmented Notch1 and HES1 production. Further investigation demonstrated that SFRP4 bound to integrin α1β1 to activate the focal adhesion kinase (FAK) pathway and that phosphorylated FAK Y397 (p-FAK Y397) aided Notch intracellular domain 1 (NICD1) nuclear translocation to form a p-FAK Y397–NICD1 complex that activated the Hes1 promoter. Taken together, the cross-talk between SFRP4, integrin α1β1, and Notch1 suppresses the cardiac differentiation of P19CL6 cells.

Secreted Frizzled-Related Protein 4 (SFRP4) is Elevated in Patients with Diabetes Mellitus.

Recently, SFRP4 was identified as a molecular link between islet inflammation and defective insulin secretion. Gene co-expression analysis detected a molecule associated with type 2 diabetes mellitus (T2D), elevated HbA1c, and reduced insulin secretion in mice as well as in a pilot sample of humans. To our knowledge SFRP4 has never been investigated in patients with different types of diabetes. We included 179 patients: 46 with type 1 diabetes (T1D), 30 age matched healthy controls for patients with T1D (CO-T1D), 55 with T2D, 37 with latent autoimmune diabetes of the adult (LADA) and 30 healthy controls (CO) for patients with T2D and LADA. Apart from anthropometric data, lipids and renal parameters were assessed. SFRP4 levels were measured by a commercial ELISA. Patients with diabetes had significant higher SFRP4 levels than CO: T2D vs. CO: 37.1±26.7 vs. 8.8±3.0 ng/ml, p<0.001; LADA vs. CO: 15.6±6.2 vs. 8.7±3.0 ng/ml, p<0.001; T1D vs. CO-T1D: 24.6±17.9 vs. 16.9±4.5 ng/ml, p=0.011. SFRP4 levels were correlated with age, BMI, HbA1c, HDL-cholesterol, and triglycerides. A multivariate model revealed HDL-cholesterol, triglycerides and BMI as predictors for SFRP4. This is the first study demonstrating that SFRP4 is significantly increased in patients with different types of diabetes suggesting that this protein is generally involved in islet dysfunction and potentially subclinical inflammation irrespective of type of diabetes.

Loss of SFRP4 Alters Body Size, Food Intake, and Energy Expenditure in Diet-Induced Obese Male Mice.

Secreted frizzled-related protein 4 (SFRP4) is an extracellular regulator of the wingless-type mouse mammary tumor virus integration site family (WNT) pathway. SFRP4 has been implicated in adipocyte dysfunction, obesity, insulin resistance, and impaired insulin secretion in patients with type 2 diabetes. However, the exact role of SFRP4 in regulating whole-body metabolism and glucose homeostasis is unknown. We show here that male Sfrp4(-/-) mice have increased spine length and gain more weight when fed a high-fat diet. The body composition and body mass per spine length of diet-induced obese Sfrp4(-/-) mice is similar to wild-type littermates, suggesting that the increase in body weight can be accounted for by their longer body size. The diet-induced obese Sfrp4(-/-) mice have reduced energy expenditure, food intake, and bone mineral density. Sfrp4(-/-) mice have normal glucose and insulin tolerance and β-cell mass. Diet-induced obese Sfrp4(-/-) and control mice show similar impairments of glucose tolerance and a 5-fold compensatory expansion of their β-cell mass. In summary, our data suggest that loss of SFRP4 alters body length and bone mineral density as well as energy expenditure and food intake. However, SFRP4 does not control glucose homeostasis and β-cell mass in mice.

Relationship between serum secreted Frizzled-related protein 4 and the pancreatic β cell function

Objective To investigate the relationship between serum secreted frizzled-related protein 4(SFRP4) and the first-phase of glucose-stimulated insulin secretion from pancreatic β cell under different glucose tolerance statuses. Methods Fifty-six patients with newly diagnosed type 2 diabetes mellitus(T2DM group), 52 patients with impaired glucose tolerance(IGT group), and 42 subjects with normal glucose tolerance(NGT group)underwent intravenous glucose tolerance test. Fasting serum SFRP4 and interleukin(IL)-1β were assayed by ELISA. Acute insulin response(AIR), the area under the curve of the first-phase(0-10 min)insulin secretion(AUC), glucose disposition index(GDI), homeostasis model assessment for β cell function index(HOMA-β), and insulin resistance index(HOMA-IR)were calculated. Results (1)The levels of SFRP4 and IL-1β in T2DM group and IGT group were significantly higher than that in NGT group[(184.38±61.34 or 141.64±40.46 or 95.46±20.13)ng/ml, P<0.01]. AIR, AUC, and GDI in T2DM group and IGT group were significantly lower than those in NGT group(P<0.01), and these results were more significantly reduced in T2DM group compared with those in IGT group.(2)SFRP4 was negatively correlated with AIR, AUC, GDI, HOMA-β(P<0.01), and positively correlated with fasting plasma glucose, 2 h plasma glucose after glucose loading, HbA1C, IL-1β, and high sensitive C-reactive protein(P<0.01). (3)Multiple stepwise regression analysis showed that AUC, HOMA-IR, and serum IL-1β level were independently associated with SFRP4. Conclusion The concentration of serum SFRP4 is closely correlated with the glycolipid metabolic disorder, the first-phase of glucose-stimulated insulin secretion, and chronic low-grade inflammation. SFRP4 may be involved in the mechanism of β cell dysfunction in type 2 diabetes mellitus. Key words: Secreted frizzled-related protein 4; β-cell function; Intravenous glucose tolerance test; Interleukin-1β; Diabetes mellitus, type 2