The osteogenic transcription factor Runx2 regulates components of the fibroblast growth factor/proteoglycan signaling axis in osteoblasts

Heparan sulfate proteoglycans cooperate with basic fibroblast growth factor (bFGF/FGF2) signaling to control osteoblast growth and differentiation, as well as metabolic functions of osteoblasts. FGF2 signaling modulates the expression and activity of Runt-related transcription factor 2 (Runx2/Cbfa1), a key regulator of osteoblast proliferation and maturation. Here, we have characterized novel Runx2 target genes in osteoprogenitors under conditions that promote growth arrest while not yet permitting sustained phenotypic maturation. Runx2 enhances expression of genes related to proteoglycan-mediated signaling, including FGF receptors (e.g., FGFR2 and FGFR3) and proteoglycans (e.g., syndecans [Sdc1, Sdc2, Sdc3], glypicans [Gpc1], versican [Vcan]). Runx2 increases expression of the glycosyltransferase Exostosin-1 (Ext1) and heparanase, as well as alters the relative expression of N-linked sulfotransferases (Ndst1 = Ndst2 > Ndst3) and enzymes mediating O-linked sulfation of heparan sulfate (Hs2st > Hs6st) or chondroitin sulfate (Cs4st > Cs6st). Runx2 cooperates with FGF2 to induce expression of Sdc4 and the sulfatase Galns, but Runx2 and FGF2 suppress Gpc6, thus suggesting intricate Runx2 and FGF2 dependent changes in proteoglycan utilization. One functional consequence of Runx2 mediated modulations in proteoglycan-related gene expression is a change in the responsiveness of bone markers to FGF2 stimulation. Runx2 and FGF2 synergistically enhance osteopontin expression (>100 fold), while FGF2 blocks Runx2 induction of alkaline phosphatase. Our data suggest that Runx2 and the FGF/proteoglycan axis may form an extracellular matrix (ECM)-related regulatory feed-back loop that controls osteoblast proliferation and execution of the osteogenic program.

Osteogenic differentiation of murine embryonic stem cells is mediated by fibroblast growth factor receptors

The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be fully elucidated. However, it has become clear that the family of fibroblast growth factors (FGFs) are centrally involved. In this study we examined the role of the FGF receptors (FGFRs 1-4) during osteogenesis in murine ES cells. Single cells were obtained after the formation of embryoid bodies, cultured on gelatin-coated plates, and coaxed to differentiate along the osteogenic lineage. Upregulation of genes was analyzed at both the transcript and protein levels using gene array, relative-quantitative PCR (RQ-PCR), and Western blotting. Deposition of a mineralized matrix was evaluated with Alizarin Red staining. An FGFR1-specific antibody was generated and used to block FGFR1 activity in mES cells during osteogenic differentiation. Upon induction of osteogenic differentiation in mES cells, all four FGFRs were clearly upregulated at both the transcript and protein levels with a number of genes known to be involved in osteogenic differentiation including bone morphogenetic proteins (BMPs), collagen I, and Runx2. Cells were also capable of depositing a mineralized matrix, confirming the commitment of these cells to the osteogenic lineage. When FGFR1 activity was blocked, a reduction in cell proliferation and a coincident upregulation of Runx2 with enhanced mineralization of cultures was observed. These results indicate that FGFRs play critical roles in cell recruitment and differentiation during the process of osteogenesis in mES cells. In particular, the data indicate that FGFR1 plays a pivotal role in osteoblast lineage determination.

Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent

Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium-signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.

Bimolecular interaction of Insulin-Like Growth Factor (IGF) binding protein-2 with αvβ3 negatively modulates IGF-I-Mediated migration and tumor growth

Both the integrin and insulin-like growth factor binding protein (IGFBP) families independently play important roles in modulating tumor cell growth and progression. We present evidence for a specific cell surface localization and a bimolecular interaction between the αvβ3 integrin and IGFBP-2. The interaction, which could be specifically perturbed using vitronectin and αvβ3 blocking antibodies, was shown to modulate IGF-mediated cellular migration responses. Moreover, this interaction was observed in vivo and correlated with reduced tumor size of the human breast cancer cells, MCF-7β3, which overexpressed the αvβ3 integrin. Collectively, these results indicate that αvβ3 and IGFBP-2 act cooperatively in a negative regulatory manner to reduce tumor growth and the migratory potential of breast cancer cells.

An acute growth factor treatment that preserves function after spinal cord contusion injury

Inflammation of the spinal cord after traumatic spinal cord injury leads to destruction of healthy tissue. This “secondary degeneration” is more damaging than the initial physical damage and is the major contributor to permanent loss of functions. In our previous study we showed that combined delivery of two growth factors, vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), significantly reduced secondary degeneration after hemi-section injury of the spinal cord in the rat. Growth factor treatment reduced the size of the lesion cavity at 30d compared to control animals and further reduced the cavity at 90d in treated animals while in control animals the lesion cavity continued to increase in size. Growth factor treatment also reduced astrogliosis and reduced macroglia/macrophage activation around the injury site. Treatment with individual growth factors alone had similar effects to control treatments. The present study investigated whether growth factor treatment would improve locomotor behaviour after spinal contusion injury, a more relevant preclinical model of spinal cord injury. The growth factors were delivered for the first 7d to the injury site via osmotic minipump. Locomotor behaviour was monitored at 1-28d after injury using the BBB score and at 30d using automated gait analysis. Treated animals had BBB scores of 18; Control animals scored 10. Treated animals had significantly reduced lesion cavities and reduced macroglia/macrophage activation around the injury site. We conclude that growth factor treatment preserved spinal cord tissues after contusion injury, thereby allowing functional recovery. This treatment has the potential to significantly reduce the severity of human spinal cord injuries.

MTI-MMP expression promotes tumor growth and angiogenesis through an up-regulation of vascular endothelial growth factor expression

Membrane type 1 metalloprotease (MT1-MMP) is a transmembrane metalloprotease that plays a major role in the extracellular matrix remodeling, directly by degrading several of its components and indirectly by activating pro-MMP2. We investigated the effects of MT1-MMP overexpression on in vitro and in vivo properties of human breast adenocarcinoma MCF7 cells, which do not express MT1-MMP or MMP-2. MT1-MMP and MMP-2 cDNAs were either transfected alone or cotransfected. All clones overexpressing MT1-MMP 1) were able to activate endogenous or exogenous pro-MMP-2, 2) displayed an enhanced in vitro invasiveness through matrigel-coated filters independent of MMP-2 transfection, 3) induced the rapid development of highly vascularized tumors when injected subcutanously in nude mice, and 4) promoted blood vessels sprouting in the rat aortic ring assay. These effects were observed in all clones overexpressing MT1-MMP regardless of MMP-2 expression levels, suggesting that the production of MMP-2 by tumor cells themselves does not play a critical role in these events. The angiogenic phenotype of MT1-MMP-producing cells was associated with an up-regulation of VEGF expression. These results emphasize the importance of MT1-MMP during tumor angiogenesis and open new opportunities for the development of antiangiogenic strategies combining inhibitors of MT1-MMP and VEGF antagonists. - Sounni, N. E., Devy, L., Hajitou, A., Frankenne, F., Munaut, C., Gilles, C., Deroanne, C., Thompson, E. W., Foidart, J. M., Noel, A. MT1-MMP expression promotes tumor growth and angiogenesis through an up-regulation of vascular endothelial growth factor expression.

Interleukin-6 is a potent inducer of S100P, which is up-regulated in androgen-refractory and metastatic prostate cancer

Elevated circulating interleukin-6 (IL6) and up-regulated S100P in prostate cancer (PCa) specimens correlate independently with progression to androgen-independent and metastatic PCa. The cause of up-regulated S100P levels in advanced PCa remains to be determined. We investigated the possibility that IL6 is an inducer of S100P. Determination of mRNA and protein levels by real-time PCR and Western blotting revealed that IL6 is a more potent inducer of S100P than the synthetic androgen, R1881, in the LNCaP/C4-2B model of PCa progression. IL6 did not require androgen to induce S100P in these cells, which express a functional androgen receptor (AR). Like R1881, IL6 was unable to induce S100P in PC3 cells that lack a functional AR. IL6 did not strongly induce the AR-dependent genes PSA and KLK2 and, contrary to R1881, down-regulated Cyr61/CCN1, a potential marker that is down-regulated in PCa. Epidermal growth factor (EGF), which like IL6 is a non-androgen activator of the AR, did not induce S100P. The data identifies a unique gene-induction profile for IL6 and suggests that IL6 may require a functional AR for S100P induction. A link between elevated IL6 and up-regulated S100P in androgen-refractory and metastatic PCa is postulated.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract (Matrigel™) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 μL collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

Time course analysis of hypoxia, granulation tissue and blood vessel growth, and remodeling in healing rat cutaneous incisional primary intention wounds

Hypoxia and the development and remodeling of blood vessels and connective tissue in granulation tissue that forms in a wound gap following full-thickness skin incision in the rat were examined as a function of time. A 1.5 cm-long incisional wound was created in rat groin skin and the opposed edges sutured together. Wounds were harvested between 3 days and 16 weeks and hypoxia, percent vascular volume, cell proliferation and apoptosis, α-smooth muscle actin, vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2, and transforming growth factor-β 1 expression in granulation tissue were then assessed. Hypoxia was evident between 3 and 7 days while maximal cell proliferation at 3 days (123.6 ± 22.2 cells/mm 2, p < 0.001 when compared with normal skin) preceded the peak percent vascular volume that occurred at 7 days (15.83 ± 1.10%, p < 0.001 when compared with normal skin). The peak in cell apoptosis occurred at 3 weeks (12.1 ± 1.3 cells/mm 2, p < 0.001 when compared with normal skin). Intense α-smooth muscle actin labeling in myofibroblasts was evident at 7 and 10 days. Vascular endothelial growth factor receptor-2 and vascular endothelial growth factor-A were detectable until 2 and 3 weeks, respectively, while transforming growth factor-β 1 protein was detectable in endothelial cells and myofibroblasts until 3-4 weeks and in the extracellular matrix for 16 weeks. Incisional wound granulation tissue largely developed within 3-7 days in the presence of hypoxia. Remodeling, marked by a decline in the percent vascular volume and increased cellular apoptosis, occurred largely in the absence of detectable hypoxia. The expression of vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2, and transforming growth factor-β 1 is evident prior, during, and after the peak of vascular volume reflecting multiple roles for these factors during wound healing.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Background: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I:IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. Methods: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. Results: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while β1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and β1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. Conclusion: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

Association between single-nucleotide polymorphisms in growth factor genes and quality of life in men with prostate cancer and the general population

Purpose Improved survival for men with prostate cancer has led to increased attention to factors influencing quality of life (QOL). As protein levels of vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) have been reported to be associated with QOL in people with cancer, we sought to identify whether single-nucleotide polymorphisms (SNPs) of these genes were associated with QOL in men with prostate cancer. Methods Multiple linear regression of two data sets (including approximately 750 men newly diagnosed with prostate cancer and 550 men from the general population) was used to investigate SNPs of VEGF and IGF-1 (10 SNPs in total) for associations with QOL (measured by the SF-36v2 health survey). Results Men with prostate cancer who carried the minor ‘T’ allele for IGF-1 SNP rs35767 had higher mean Role-Physical scale scores (≥0.3 SD) compared to non-carriers (p < 0.05). While this association was not identified in men from the general population, one IGF-1 SNP rs7965399 was associated with higher mean Bodily Pain scale scores in men from the general population that was not found in men with prostate cancer. Men from the general population who carried the rare ‘C’ allele had higher mean Bodily Pain scale scores (≥0.3 SD) than non-carriers (p < 0.05). Conclusions Through identifying SNPs that are associated with QOL in men with prostate cancer and men from the general population, this study adds to the mapping of complex interrelationships that influence QOL and suggests a role for IGF-I in physical QOL outcomes. Future research may identify biomarkers associated with increased risk of poor QOL that could assist in the provision of pre-emptive support for those identified at risk.

The chromosome 16q region associated with ankylosing spondylitis includes the candidate gene tumour necrosis factor receptor type 1-associated death domain (TRADD)

Objective: To replicate and refine the reported association of ankylosing spondylitis (AS) with two nonsynonymous single nucleotide polymorphisms (nsSNPs) on chromosome 16q22.1. Methods: Firstly, 730 independent UK patients with AS were genotyped for rs9939768 and rs6979 and allele frequencies were compared with 2879 previously typed historic disease controls. Secondly, the two data sets were combined in meta-analyses. Finally, 5 tagging SNPs, located between rs9939768 and rs6979, were analysed in 1604 cases and 1020 controls. Results: The association of rs6979 with AS was replicated, p=0.03, OR=1.14 (95% CI 1.01 to 1.28), and a trend for association with rs9939768 detected, p=0.06, OR=1.25 (95% CI 0.99 to 1.57). Meta-analyses revealed association of both SNPs with AS, p=0.0008, OR=1.31 (95% CI 1.12 to 1.54) and p=0.0009, OR=1.15 (95% CI 1.06 to 1.23) for rs9939768 and rs6979, respectively. New associations with rs9033 and rs868213 (p=0.00002, OR=1.23 (95% CI 1.12 to 1.36) and p=0.00002 OR=1.45 (95% CI 1.22 to 1.72), respectively, were identified. Conclusions: The region on chromosome 16 that has been replicated in the present work is interesting as the highly plausible candidate gene, tumour necrosis factor receptor type 1 (TNFR1)-associated death domain (TRADD), is located between rs9033 and rs868213. It will require additional work to identify the primary genetic association(s) with AS.

Evidence of genetic association between TNFRSF1A encoding the p55 tumour necrosis factor receptor, and ankylosing spondylitis in UK Caucasians

Objectives: To replicate the possible genetic association between ankylosing spondylitis (AS) and TNFRSF1A. Methods: TNFRSF1A was re-sequenced in 48 individuals with AS to identify novel polymorphisms. Nine single nucleotide polymorphisms (SNPs) in TNFRSF1A and 5 SNPs in the neighbouring gene SCNN1A were genotyped in 1604 UK Caucasian individuals with AS and 1019 matched controls. An extended study was implemented using additional genotype data on 8 of these SNPs from 1400 historical controls from the 1958 British Birth Cohort. A meta-analysis of previously published results was also undertaken. Results: One novel variant in intron 6 was identified but no new coding variants. No definite associations were seen in the initial study but in the extended study there were weak associations with rs4149576 (p=0.04) and rs4149577 (p=0.007). In the metaanalysis consistent, somewhat stronger associations were seen with rs4149577 (p=0.002) and rs4149578 (p=0.006). Conclusions: These studies confirm the weak genetic associations between AS and TNFRSF1A. In view of the previously reported associations of TNFRSF1A with AS, in Caucasians and Chinese, and the biological plausibility of this candidate gene, replication of this finding in well powered studies is clearly indicated.

The effect of transforming growth factor β1 gene polymorphisms in ankylosing spondylitis

Objectives. To determine whether genetic polymorphisms in or near the transforming growth factor β1 (TGFB1) locus were associated d with susceptibility to or severity of ankylosing spondylitis (AS). Methods. Five intragenic single-nucleotide polymorphisms (SNP) and three microsatellite markers flanking the TGFB1 locus were genotyped. Seven hundred and sixty-two individuals from 184 multiplex families were genotyped for the microsatellite markers and two of the promoter SNPs. One thousand and two individuals from 212 English and 170 Finnish families with AS were genotyped for all five intragenic SNPs. A structured questionnaire was used to assess the age of symptom onset, disease duration and disease severity scores, including the BASDAI (Bath Ankylosing Spondylitis Disease Activity Index) and BASFI (Bath Ankylosing Spondylitis Functional Index). Results. A weak association was noted between the rare TGFB1 + 1632 T allele and AS in the Finnish population (P = 0.04) and in the combined data set (P = 0.03). No association was noted between any other SNPs or SNP haplotype and AS, even among those families with positive non-parametric linkage scores. The TGFB1 +1632 polymorphism was also associated with a younger age of symptom onset (English population, allele 2 associated with age of onset greater by 4.2 yr, P = 0.05; combined data set, allele 2 associated with age of onset greater by 3.2 yr, P = 0.02). A haplotype of coding region SNPs (TGFB1 +869/ +915+1632 alleles 2/1/2) was associated with age of symptom onset in both the English parent-case trios and the combined data set (English data set, haplotype 2/1/2 associated with age of onset greater by 4.9 yr, P = 0.03; combined data set, haplotype 2/1/2 associated with greater age of onset by 4.2 yr, P = 0.006). Weak linkage with AS susceptibility was noted and the peak LOD score was 1.3 at distance 2 cM centromeric to the TGFB1 gene. No other linkage or association was found between quantitative traits and the markers. Conclusion. This study suggests that the polymorphisms within the TGFB1 gene play at most a small role in AS and that other genes encoded on chromosome 19 are involved in susceptibility to the disease.