Molecular cloning and expression profiling of insulin-like growth factor 2 and IGF-binding protein 6 in Clarias magur (Hamilton 1822).

Growth is an important trait in aquaculture and the major genes that regulate it are Insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs). In this study, the full-length coding sequences of IGF2 and IGFBP6 genes in the Indian catfish Clarias magur were cloned and characterized. The full-length cDNA sequences of IGF2 and IGFBP6 were 885 bp (ORF 642 bp) and 928 bp (ORF 600 bp), encoding 213 and 199 amino acids, respectively. Bioinformatics analyses revealed that the magur IGF2 and IGFBP6 proteins are hydrophilic and secretory in nature. Sequence alignment with other teleosts and mammalian orthologues shows conservation of the functional domains. Gene expression analysis in 6 individuals each of high (298 ± 5.0 g) and low (210 ± 6.0 g) growth performing families showed significantly (p < 0.05) higher expression (2.5-3 fold) of IGF2, and lower expression (∼2.5 fold) of IGFBP6 in liver and muscle of fast-growing fish. This study suggests that IGF2 could be playing a major role in the growth regulation of magur. These genes and their expression patterns could be developed into growth-associated markers for magur and other catfishes.

Effect of IGFBP6 Knockdown on Proteins Regulating Exosome Synthesis and Secretion in MDA-MB-231 Cell Line.

One of the potential causes of cancer recurrence is disruption of the cell-cell communication in the primary tumors that is realized, among other things, through secretion and uptake of exosomes by cells. Low expression of the IGFBP6 gene (insulin-like growth factor binding protein 6) is associated with a high recurrence rate and can serve as a prognostic marker of luminal breast cancer. The knockdown of the IGFBP6 gene leads to significant changes in lipid metabolism. We performed a quantitative analysis of both exosomes and proteins involved in the mechanism of their biogenesis. Changes in the expression profile of mRNAs and their proteins responsible for the synthesis and secretion of exosomes were revealed. We showed a decrease in the expression of the of the VPS28 gene mRNA (vacuolar protein sorting-associated protein 28) and the corresponding protein by 2.3 and 5.6 times, respectively. The secretion of exosomes by MDA-MB-231 cells with IGFBP6 knockdown decreased by 2 times. We discussed a mechanism of disruption of cell-cell communication.

Extensive serum biomarker analysis in patients with non-small-cell lung carcinoma.

Lung cancer is a common malignant disease, nearly 2.09 million new patients occurred last year. Approximately 85% of the patients are classified as non-small-cell lung cancer (NSCLC). It is therefore important to identify new diagnostic and prognostic biomarkers for the early detection of this disease. The presented study identifies biomarkers in the serum of NSCLC patients. The expression of 274 cytokines was measured by a novel antibody array methodology and ELISA was applied to validate the array results. The levels of MIP-1 α, IL-8, MIP-1 ß, Resistin, GDF-15, HGF, CA125, FLRG, VCAM-1, DKK-3, sTNF-R1, CTACK, Acrp30, CXCL-16 and LYVE-1 were significantly higher in serum from NSCLC patients, while the level of TIMP-2 and IGFBP-6 were lower. More importantly, the validation supported the result of the antibody array. The result of the antibody array indicates that these cytokines might be novel auxiliary biomarkers in the diagnosis and prognosis of NSCLC.

Paracrine recruitment and activation of fibroblasts by c-Myc expressing breast epithelial cells through the IGFs/IGF-1R axis.

Fibroblasts are among the most abundant stromal cells in the tumor microenvironment (TME), progressively differentiating into activated, motile, myofibroblast-like, protumorigenic cells referred to as Cancer-Associated Fibroblasts (CAFs). To investigate the mechanisms by which epithelial cells direct this transition, the early stages of tumorigenesis were exemplified by indirect cocultures of WI-38 or human primary breast cancer fibroblasts with human mammary epithelial cells expressing an inducible c-Myc oncogene (MCF10A-MycER). After c-Myc activation, the conditioned medium (CM) of MCF10A-MycER cells significantly enhanced fibroblast activation and mobilization. As this was accompanied by decreased insulin-like growth factor binding protein-6 (IGFBP-6) and increased insulin-like growth factor-1 and IGF-II (IGF-I, IGF-II) in the CM, IGFs were investigated as key chemotactic factors. Silencing IGFBP-6 or IGF-I or IGF-II expression in epithelial cells or blocking Insulin-like growth factor 1 receptor (IGF-1R) activity on fibroblasts significantly altered fibroblast mobilization. Exposure of WI-38 fibroblasts to CM from induced MCF10A-MycER cells or to IGF-II upregulated FAK phosphorylation on Tyr397 , as well as the expression of α-smooth muscle actin (α-SMA), features associated with CAF phenotype and increased cell migratory/invasive behavior. In three-dimensional (3D)-organotypic assays, WI-38 or human primary fibroblasts, preactivated with either CM from MCF10A-MycER cells or IGFs, resulted in a permissive TME that enabled nontransformed MCF10A matrix invasion. This effect was abolished by inhibiting IGF-1R activity. Thus, breast epithelial cell oncogenic activation and stromal fibroblast transition to CAFs are linked through the IGFs/IGF-1R axis, which directly promotes TME remodeling and increases tumor invasion.

Insulin-like growth factor binding protein (IGFBP6) is a cross-species tendon marker.

The main challenge in tendon injury management is suboptimal tissue healing that fails to re-establish original tendon function. Tissue bioengineering is a promising approach for tendon therapy, with potential to improve its functional outcomes. However, evaluation criteria for tissue-engineered tendon are unclear due to the lack of specific markers of differentiated tendon. The study aim was to identify a panel of genes that characterised tendons in comparison to cartilage or muscles and validate those genes, both in human and key species used as models for tendon diseases. Gene expression profiling of rat tendon and cartilage in whole-tissue samples and primary tenocytes and chondrocytes was undertaken using two independent microarray platforms. Genes that demonstrated high expression correlation across two assays were validated by qRT-PCR in rat tendon relative to cartilage and muscle. Five genes demonstrating the highest tendon-related expression in the validation experiment (ASPN, ECM1, IGFBP6, TNMD, THBS4) were further evaluated by qRT-PCR in ovine, equine and human tissue. The group of tendon markers, identified by unbiased transcriptomic analysis of rat musculoskeletal tissues, demonstrated species-dependent profiles of expression. Insulin-like growth factor binding protein 6 (IGFBP6) was identified as the only universal tendon marker. Further investigation in equine tendon showed that IGFBP6 expression was not affected by ageing or tendon function but decreased in anatomical regions subjected to elevated compressive force. IGFBP6 is a robust cross-species marker of tendon phenotype and may find application in evaluation of tendon physiology and guided differentiation of permissive cells towards functional tenocytes.

From fever to immunity: A new role for IGFBP-6?

Fever is a fundamental response to infection and a hallmark of inflammatory disease, which has been conserved and shaped through millions of years of natural selection. Although fever is able to stimulate both innate and adaptive immune responses, the very nature of all the molecular thermosensors, the timing and the detailed mechanisms translating a physical trigger into a fundamental biological response are incompletely understood. Here we discuss the consequence of hyperthermic stress in dendritic cells (DCs), and how the sole physical input is sensed as an alert stimulus triggering a complex transition in a very narrow temporal window. Importantly, we review recent findings demonstrating the significant and specific changes discovered in gene expression and in the metabolic phenotype associated with hyperthermia in DCs. Furthermore, we discuss the results that support a model based on a thermally induced autocrine signalling, which rewires and sets a metabolism checkpoint linked to immune activation of dendritic cells. Importantly, in this context, we highlight the novel regulatory functions discovered for IGFBP-6 protein: induction of chemotaxis; capacity to increase oxidative burst and degranulation of neutrophils, ability to induce metabolic changes in DCs. Finally, we discuss the role of IGFBP-6 in autoimmune disease and how novel mechanistic insights could lead to exploit thermal stress-related mechanisms in the context of cancer therapy.

Role of IGFBP6 Protein in the Regulation of Epithelial-Mesenchymal Transition Genes.

Protein IGFBP6 plays an important role in the pathogenesis of many malignant tumors, including breast cancer. The relationship between IGFBP6 protein and the expression of genes associated with the epithelial-mesenchymal transition is studied. Gene IGFBP6 knockdown does not trigger the epithelial-mesenchymal transition in MDA-MB-231 cells, but modifies significantly the expression of many genes involved in this process. A decrease of IGFBP6 expression can involve a decrease in the expression of N-cadherin and transcription factor Slug.

Trophic factors from adipose tissue-derived multi-lineage progenitor cells promote cytodifferentiation of periodontal ligament cells.

Stem and progenitor cells are currently being investigated for their applicability in cell-based therapy for periodontal tissue regeneration. We recently demonstrated that the transplantation of adipose tissue-derived multi-lineage progenitor cells (ADMPCs) enhances periodontal tissue regeneration in beagle dogs. However, the molecular mechanisms by which transplanted ADMPCs induce periodontal tissue regeneration remain to be elucidated. In this study, trophic factors released by ADMPCs were examined for their paracrine effects on human periodontal ligament cell (HPDL) function. ADMPC conditioned medium (ADMPC-CM) up-regulated osteoblastic gene expression, alkaline phosphatase activity and calcified nodule formation in HPDLs, but did not significantly affect their proliferative response. ADMPCs secreted a number of growth factors, including insulin-like growth factor binding protein 6 (IGFBP6), hepatocyte growth factor and vascular endothelial growth factor. Among these, IGFBP6 was most highly expressed. Interestingly, the positive effects of ADMPC-CM on HPDL differentiation were significantly suppressed by transfecting ADMPCs with IGFBP6 siRNA. Our results suggest that ADMPCs transplanted into a defect in periodontal tissue release trophic factors that can stimulate the differentiation of HPDLs to mineralized tissue-forming cells, such as osteoblasts and cementoblasts. IGFBP6 may play crucial roles in ADMPC-induced periodontal regeneration.

Linkage analysis of SNPs in IGFBP-6 and its relation with the body sizes of pig.

Insulin-like growth factor binding protein-6 (IGFBP-6) is a member of the IGFBP family, which is known to be a key factor in regulating the effect of insulin-like growth factor-2 (IGF-2) on the animal growth and development. Gene sequences of 3'-untranslated regions (UTR) and exon 4 of IGFBP-6 may influence the expression and proteolysis of IGFBP-6. In this study, 551 bp of the IGFBP-6 (including 257 bp of intron 3, exon 4, and 170 bp of 3' UTR) were sequenced and compared in the Bama and Tibetan mini-pigs, the Landrace and Large White pigs, and the Northeast wild boars. Six single nucleotide polymorphisms (SNPs) were detected in the IGFBP-6, in which T593C, T636C, and T745C were in intron 3, A67G was in exon 4, and G37A was in 3' UTR. T636C, T745C, and A67G were in linkage and formed four kinds of haplotypes, with CCT being the dominant haplotype in the mini-pigs; however, the haplotype block was not formed in the Landrace pigs and Large White pigs or the Northeast wild boars. Based on the above results, we concluded that the SNPs and haplotype of the IGFBP-6 may be related to the mini-size formation of the pig.

Prohibitin-2 binding modulates insulin-like growth factor-binding protein-6 (IGFBP-6)-induced rhabdomyosarcoma cell migration.

Insulin-like growth factor (IGF)-binding protein (IGFBP)-6 decreases cancer cell proliferation and survival by inhibiting the effects of IGF-II. More recently, IGFBP-6 was found to promote the migration of rhabdomyosarcoma (RMS) cells in an IGF-independent manner, and MAPK pathways were involved in this process. However, the precise molecular mechanisms of these IGF-independent migratory actions of IGFBP-6 are largely unknown. Here, we report that prohibitin-2 (PHB2), a single-span membrane protein, is a key regulator of IGFBP-6-induced RMS cell migration. PHB2 and IGFBP-6 co-localize on the RMS cell surface, and they specifically interact, as demonstrated by affinity chromatography, co-immunoprecipitation, biosensor analysis, and confocal microscopy. Binding affinities for PHB2 are 9.0 ± 1.0 nM for IGFBP-6 and 10.2 ± 0.5 nM for mIGFBP-6, a non-IGF-binding mutant of IGFBP-6. The C-domain but not the N-domain of IGFBP-6 is involved in PHB2 binding. In addition, IGFBP-6 indirectly increases PHB2 tyrosine phosphorylation on RMS membranes. Importantly, PHB2 knockdown completely abolished IGFBP-6-mediated RMS cell migration. In contrast, IGFBP-6-induced MAPK pathway activation was not affected, suggesting that PHB2 may act as a downstream effector of these pathways. These results indicate that PHB2 plays a key role in this IGF-independent action of IGFBP-6 and suggest a possible therapeutic target for RMS.

Expression levels of mRNA for insulin-like growth factors 1 and 2, IGF receptors and IGF binding proteins in in vivo and in vitro grown bovine follicles.

This study investigated mRNA levels for insulin-like growth factors (IGFs) IGF1 (IGF-I) and IGF2 (IGF-II), IGF receptors (IGF1R and IGF2R), and binding proteins (IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6) in bovine follicles of 0.2, 0.5 or 1.0 mm in diameter. mRNA expression levels in in vitro cultured follicles that reached approximately 0.5 mm were compared with that of in vivo grown follicles. IGF1R and IGF2R expression levels in 0.5 mm in vivo follicles were higher than in 1.0 or 0.2 mm follicles, respectively. IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6 showed variable expression in the follicular size classes analyzed. In vitro grown follicles had significantly reduced expression levels for IGF1, IGF1R, IGFBP-3, IGFBP-5 and IGFBP-6 mRNA when compared with 0.2 mm follicles, but, when compared with in vivo grown follicles (0.5 mm), only IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 showed a reduction in their expression. In conclusion, IGFs, their receptors and IGFBPs showed variable expression of mRNA levels in the follicular size classes analyzed.

IGF binding protein-6 expression in vascular endothelial cells is induced by hypoxia and plays a negative role in tumor angiogenesis.

Hypoxia stimulates tumor angiogenesis by inducing the expression of angiogenic molecules. The negative regulators of this process, however, are not well understood. Here, we report that hypoxia induced the expression of insulin-like growth factor binding protein-6 (IGFBP-6), a tumor repressor, in human and rodent vascular endothelial cells (VECs) via a hypoxia-inducible factor (HIF)-mediated mechanism. Addition of human IGFBP-6 to cultured human VECs inhibited angiogenesis in vitro. An IGFBP-6 mutant with at least 10,000-fold lower binding affinity for IGFs was an equally potent inhibitor of angiogenesis, suggesting that this action of IGFBP-6 is IGF-independent. The functional relationship between IGFBP-6 and vascular endothelial growth factor (VEGF), a major hypoxia-inducible angiogenic molecule, was examined. While VEGF alone increased angiogenesis in vitro, co-incubation with IGFBP-6 abolished VEGF-stimulated angiogenesis. The in vivo role of IGFBP-6 in angiogenesis was tested in flk1:GFP zebrafish embryos, which exhibit green fluorescence protein in developing vascular endothelium, permitting visualization of developing blood vessels. Injection of human IGFBP-6 mRNA reduced the number of embryonic inter-segmental blood vessels by ∼40%. This anti-angiogenic activity is conserved in zebrafish because expression of zebrafish IGFBP-6b had similar effects. To determine the anti-angiogenic effect of IGFBP-6 in a tumor model, human Rh30 rhabdomyosarcoma cells stably transfected with IGFBP-6 were inoculated into athymic BALB/c nude mice. Vessel density was 52% lower in IGFBP-6-transfected xenografts than in vector control xenografts. These results suggest that the expression of IGFBP-6 in VECs is up-regulated by hypoxia and IGFBP-6 inhibits angiogenesis in vitro and in vivo.

The clinical characteristics and prognostic value of IGFBP6 in glioma.

BACKGROUND: Glioma is the most common intrinsic tumor in central nervous system and is characterized by their diffuse infiltration of the brain tissue. Insulin-like Growth Factor Binding Protein-6 (IGFBP6) was associated with the insulin-like growth factor binding and insulin-like growth factor II binding processes in many cancers. Herein, we aimed to investigate the biological functions and clinical features of IGFBP6 in gliomas. METHODS: Totally, we collected 325 RNA sequencing data from CGGA dataset as training cohort, and 969 RNA sequencing data from TCGA dataset as validation cohort. The clinical and molecular characteristics analysis and gene ontology analysis of IGFBP6 were performed. All analyses and graphs were produced based on R language. RESULTS: We found that IGFBP6 expression was significantly upregulated in GBM patients and downregulated in IDH mutant patients. Receiver Operating Characteristic (ROC) analysis revealed that IGFBP6 could be used as a biomarker to predict TCGA mesenchymal subtype. GO analysis revealed that IGFBP6 was correlated with immunological functions and inflammation activities. Meanwhile, higher expression of IGFBP6 suggested significant relationship with worse prognosis in glioma patients. CONCLUSIONS: Our findings improved the understanding of IGFBP6 in glioma, and IGFBP6 might be a potential therapeutic target for glioma patients in future clinical trials.

IGFBP-6 plays a role as an oncosuppressor gene in NPC pathogenesis through regulating EGR-1 expression.

Nasopharyngeal carcinoma (NPC) is prevalent in south-eastern Asia, particularly southern China, Singapore and Taiwan. The aim of this study was to identify the pivotal genes that may be altered during NPC progression. Using cDNA microarray analysis, we compared the expression of 18 genes between NPC and normal nasomucosal cells. qRT-PCR analysis found the expression of IBFBP-6 in NPC cell lines and immunolocalization of IGFBP-6 in NPC to be very weak. To explore the effects of IGFBP-6 on NPC tumour growth, we constructed inducible plasmids containing full-length IGFBP-6 cDNA (pBIG2i-IGFBP-6) and established pBIG2i-IGFBP-6-transfected NPC stable cell lines (NPC-TW01-pBIG2i-IGFBP-6). We then performed functional analysis of the IGFBP-6 in cell lines in vitro and in vivo. Over-expression of IGFBP-6 significantly suppressed the proliferation, invasion and metastatic activity of NPC cells and increased their apoptosis. We found the EGR-1, caspase-1 and TSP-1 genes to be markedly up-regulated when NPC-pBIG2i-IGFBP-6 was treated with doxycycline. Knocking down EGR-1 with EGR-1 siRNA resulted in a decrease in expression of caspase-1, TSP-1 and EGR-1 but not the expression of IGFBP-6. However, in knockdown cells the unchanged expression of IGFBP-6 did not inhibit the migration of NPC cells. Chromatin immunoprecipitation and luciferase reporter assay experiments showed that IGFBP-6 bound the EGR-1 promoter regions and activated EGR-1 promoter. We conclude that IGFBP-6 can regulate the progression of NPC by regulating the expression of EGR-1. These results suggest that IGFBP-6 could be used as a new target in NPC therapy.

Cyclopamine blocked the growth of colorectal cancer SW116 cells by modulating some target genes of Gli1 in vitro.

BACKGROUND/AIMS: Hedgehog (Hh) pathway has been considered as a therapy target for various cancer entities. However, its mechanism in colorectal cancer is still unclear. METHODOLOGY: We analyzed the expression of Hh pathway members in colorectal adenomas and cancer cell lines and then studied its relationship with survival of colorectal cancer cells through inhibiting Hh pathway by cyclopamine. Moreover, we studied the regulation of Gli1 on insulin-like growth factor binding protein 6 (IGFBP6) and B-cell CLL/lymphoma 2 (Bcl-2) genes at the level of transcription by XChIP and cyclopamine inhibition assay. RESULTS: Sonic hedgehog (Shh), Smoothened (Smo), patched (Ptch) and Gli1 genes mRNA were expressed in SW116 cells. Gli1 bound to promoter regions of Bcl-2 and IGFBP6 genes, cyclopamine inhibited proliferation and induced apoptosis through inhibiting the transcriptions of IGFBP6 (p=0.003), proliferating cell nuclear antigen (PCNA) (p=0.014) and Bcl-2 (p=0.013), and increasing that of BCL2-associated X protein (Bax) and BCL2-antagonist/killer 1 (Bak1) (p=0.003 and 0.001, respectively) in SW116 cells. CONCLUSIONS: Hh pathway is aberrant activation in part colorectal carcinomas cell lines and its inhibitor may be an effectual agent for colorectal cancer chemoprevention. It may be one of the mechanisms that Gli1 maintained cell survival by binding the promoter regions and facilitating transcription of IGFBP6 and Bcl-2 genes.

A regulative epigenetic circuit supervised by HDAC7 represses IGFBP6 and IGFBP7 expression to sustain mammary stemness.

Background: In the breast, the pleiotropic epigenetic regulator HDAC7 can influence stemness. Materials & Methods: The authors used MCF10 cells knocked-out for HDAC7 to explore the contribution of HDAC7 to IGF1 signaling. Results: HDAC7 buffers H3K27ac levels at the IGFBP6 and IGFBP7 genomic loci and influences their expression. In this manner, HDAC7 can tune IGF1 signaling to sustain stemness. In HDAC7 knocked-out cells, RXRA promotes the upregulation of IGFBP6/7 mRNAs. By contrast, HDAC7 increases FABP5 expression, possibly through repression of miR-218. High levels of FABP5 can reduce the delivery of all-trans-retinoic acid to RXRA. Accordingly, the silencing of FABP5 increases IGFBP6 and IGFBP7 expression and reduces mammosphere generation. Conclusion: The authors propose that HDAC7 controls the uptake of all-trans-retinoic acid, thus influencing RXRA activity and IGF1 signaling.

[Correlation of the methylation status of CpG islands in the promoter region of 10 genes with the 5-Fu chemosensitivity in 3 breast cancer cell lines].

OBJECTIVE: To explore the relationship between the methylation status of CpG islands in the promoter region of 10 genes in breast cancer cells and their sensitivity to 5-fluouracil (5-Fu), and to identify the genes responsible for the 5-Fu resistance in breast cancer. METHODS: Three cell lines (differently resistant to chemotherapy) were used in this study: Bcap-37 (IC(50): 289.77 microg/ml), T47D (IC(50): 134.16 microg/ml) and ZR-75-30 (IC(50): 4.20 microg/ml). The methylation profile of 10 genes (BAG1, C11ORF31, CBR1, CBR4, GJA1, FOXL2, IGFBP6, P4HA1, SRI and TYMS) in the 3 breast cancer cell lines was determined by methylation specific PCR. The steady-state mRNAs of ABCC8, CHFR and IGFBP6 genes were quantified by real-time RT PCR analysis. RESULTS: Among the 10 genes, only genes IGFBP6 and FOXL2 displayed differential DNA methylation pattern between the 5-Fu-resistant and 5-Fu-sensitive cell lines. The mRNA expression level of genes PRSS21, LOX, IGFBP6, ABCC8 and CHFR was quantified by real-time RT-PCR analysis. Except for CHFR, the expression level of the other 4 genes was correlated with the methylation status of CpG islands, namely, a lower expression level with methylation status and a higher level with demethylation status. CONCLUSION: The results of the present study have demonstrated that there are 8 genes with differential methylation status in chemosensitive and chemoresistant breast cancer cell lines, i.e. two genes more than the six genes we reported previously. Our findings provide both mechanistic insights for the drug resistance of breast cancer and the basis for further studies on potential application of the DNA methylation in this set of genes for prediction of chemosensitivity of breast cancer.

Guar gum different from Ganoderma lucidum polysaccharide in alleviating colorectal cancer based on omics analysis.

It is unclear if guar gum can alleviate colorectal cancer (CRC). We evaluated the effect of guar gum (unmodified) on the mortality, colon status, serous tumor necrosis factor-alpha (TNF-α) concentration, and gut microbial and colonic epithelial cell gene expression profiles in CRC mice and performed omics analyses to compare these with those of Ganoderma lucidum polysaccharide (GLP), whose main component is ß-glucan (>90%). We found that guar gum had a CRC alleviating effect. However, it showed a 20% higher mortality rate, shorter colon length, worse colon status, larger number and size of tumors, higher concentration of serous TNF-α and upregulation of epithelial cell genes (Il10, Cytl1, Igkv7-33, Ighv1-14, Igfbp6 and Foxd3) compared to that of GLP. The higher relative abundance of Akkermansia, the alteration of microbial metabolic pathways, especially those involving chaperones and folding catalysts, fatty acid biosynthesis, glycerophospholipid metabolism, glycolysis/gluconeogenesis, lipid biosynthesis and pyruvate metabolism, and the upregulation of specific genes (Mcpt2, Mcpt9, Des and Sostdc1) were also determined in animals fed a guar gum diet. The results suggested that the alleviating effect of guar gum (an inexpensive polysaccharide) on CRC was inferior to that of GLP (a more expensive polysaccharide). This could potentially be attributed to the increased presence of Akkermansia, the alteration of 10 microbial metabolic pathways and the upregulation of 4 epithelial cell genes.

Ruminal epithelial insulin-like growth factor-binding proteins 2, 3, and 6 are associated with epithelial cell proliferation.

The aim of this study was to identify factors that regulate ruminal epithelial insulin-like growth factor-binding protein (IGFBP) expression and determine its role in rumen epithelial cell proliferation. Primary bovine rumen epithelial cells (BREC) were incubated with short-chain fatty acids (SCFAs) at pH 7.4 or 5.6, lactate, lipopolysaccharide (LPS), insulin-like growth factor-I (IGF-I), -II (IGF-II), or recombinant bovine IGFBP2 (rbIGFBP2). The mRNA expression levels of IGFBP in BREC were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation rate of BREC was analyzed using a WST-1 assay. IGFBP2 gene expression tended to be lower with SCFA treatment (p < .1), and IGFBP6 gene expression was significantly lower with SCFA treatment (p < .05). IGFBP3 and IGFBP6 gene expression tended to be higher with d-Lactate treatment (p < .1). IGFBP3 gene expression was significantly higher (p < .05) with LPS treatment. BREC treated with IGF-I grew more rapidly than vehicle control-treated cells (p < .01); however, recombinant bovine rbIGFBP2 inhibited IGF-I-induced proliferation. IGF-II and/or rbIGFBP2 did not affect BREC proliferation. Taken together, SCFA treatment decreased IGFBP2 and IGFBP6 expression in rumen epithelial cells, and lower expression of these IGFBP might promote rumen epithelial cell proliferation by facilitating IGF-I.

Transcriptional activation of insulin-like growth factor binding protein 6 by 17beta-estradiol in SaOS-2 cells.

Osteoblasts can synthesize the insulin-like growth factors (IGFs) and the IGF-binding proteins (IGFBPs), which may either enhance or attenuate IGF-stimulated bone cell proliferation. Since estrogen induced osteoblastic differentiation and proliferation through an estrogen-responsive gene in target cells, we investigated the effects of estrogen on IGFBP-6 expression in the human osteoblastic-like cell line SaOS-2. Expressions of IGFBP-6 protein and mRNA increased 2.8 and 2-fold, respectively, in the presence of 17-beta-estradiol (E2) (0.01 to 1 micronM) and estrogen receptor (ER) in SaOS-2 cells. On the other hand, E2 induced a 2-fold increase in SaOS-2 cell proliferation. To identify genomic sequences associated with estrogen responsiveness, the 5'-promoter region (-44 to +118) of the IGFBP-6 gene was cloned into a chloramphenicol acetyltransferase (CAT) reporter vector. E2 induced a 3-fold increase in CAT activity in SaOS-2 cells transiently transfected with this construct. Identification of the estrogen-responsive element (ERE) [5-CCTTCA CCTG-3] (-9 to +1) in this IGFBP-6 gene promoter region was confirmed using electromobility shift assays and deletion analysis. This functional ERE was important for E2-induced trans-activation of the IGFBP-6 gene. These results demonstrate that E2 exhibits a positive effect on IGFBP-6 gene transcription through estrogen-liganded ER binding to the functional ERE in the IGFBP-6 gene promoter in SaOS-2 cells.