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At least 19 recordsLinked to original sources
PubMed2001

Bone sialoprotein, bone morphogenetic protein 6 and thymidine phosphorylase expression in localized human prostatic adenocarcinoma as predictors of clinical outcome: a clinicopathological and immunohistochemical study of 43 cases.

PURPOSE: Skeletal metastases are the hallmark of advanced prostate cancer and recurrence after local surgery is common. Currently to our knowledge no biological markers predict the risk of disease progression in individuals with localized prostate cancer. In a search for predictive markers we evaluated the expression of bone sialoprotein and bone morphogenetic protein 6, 2 bone related proteins, and the angiogenic factor thymidine phosphorylase. MATERIALS AND METHODS: The study population included 43 men who presented with localized prostate cancer treated with radical prostatectomy. Bone sialoprotein, bone morphogenetic protein 6 and thymidine phosphorylase expression was assessed by immunohistochemical testing. Results were analyzed in relation to pathological disease stage, Gleason score and clinical outcome. Clinical followup was 4.3 to 11.4 years after surgery (median 7.9). RESULTS: Disease did not progress in 17 of the 43 cases, while recurrence and/or metastasis developed in the other 26 at a median of 6.5 and 6.9 years, respectively. Bone sialoprotein and bone morphogenetic protein 6 expression detected in 28 (65%) and 29 (67%) of the 43 samples, respectively, was significantly associated (p = 0.0001). Thymidine phosphorylase detected in 26 samples (60%) was not related to bone sialoprotein and/or bone morphogenetic protein 6 positivity. Bone sialoprotein and/or bone morphogenetic protein 6 expression correlated with bone metastasis, while thymidine phosphorylase expression was related to local recurrence (p = 0.002 and/or 0.007, and 0.00007, respectively). On multivariate analysis only the correlation of thymidine phosphorylase expression with recurrence remained statistically significant (p = 0.002). Co-expression of the 3 markers was observed in the samples of 10 of the 11 patients (90%) with bone metastases and only in 5 of the 17 (29%) who were disease-free. CONCLUSIONS: This study indicates that the expression of bone sialoprotein, bone morphogenetic protein 6 and thymidine phosphorylase determined at a clinically early stage of disease by a simple immunohistochemical technique would enable subgroups of patients to be identified that are at different risks of bone metastasis or recurrence. Detection of such markers would provide additional prognostic information that would be useful for patients with intermediate or low Gleason score or stage disease. These patients would benefit from a more adapted clinical follow-up.

Adenocarcinoma↗
PubMed2006

Myofibroblast progenitor cells are increased in number in patients with type 1 diabetes and express less bone morphogenetic protein 6: a novel clue to adverse tissue remodelling?

AIMS/HYPOTHESIS: Growth factor imbalance and endothelial progenitor cell dysfunction are well-known elements of the inappropriate response to injury in human and experimental diabetes. We hypothesised that in diabetes the outgrowth of myofibroblast progenitor cells (MFPCs) is also altered and that this relates to aberrant gene expression of growth factors involving members of the TGF-beta/bone morphogenetic protein (BMP) superfamily. SUBJECTS AND METHODS: MFPCs were cultured from peripheral blood mononuclear cells of patients with type 1 diabetes and control subjects. Microarray analysis, quantitative PCR and ELISA were used to identify differentially regulated TGF-beta/BMP superfamily genes in diabetes- and control-derived MFPC. Possible effects of BMP6 on TGF-beta-induced gene expression were examined in cultured renal fibroblasts (TK173 cells). RESULTS: Blood from diabetic patients yielded higher numbers of MFPCs than blood from control subjects (1.6-fold increase, p<0.05), involving increased proliferation and decreased apoptosis. BMP6 mRNA and protein were downregulated in MFPCs derived from patients with diabetes (3.9- and 1.8-fold decrease, respectively, p<0.05). Furthermore, an inverse correlation was observed between BMP6 mRNA level and the number of MFPCs in patients with diabetes (r=-0.85, p<0.05). In TK173 cells, BMP6 antagonised the TGF-beta-induced expression of the genes encoding plasminogen activator inhibitor-1 and connective tissue growth factor (70 and 50% reduction, respectively). CONCLUSIONS/INTERPRETATION: Considering the importance of BMP6 in processes such as angiogenesis and its novel anti-TGF-beta effects, we propose that the excess numbers of BMP6-deficient MFPCs may favour adverse tissue remodelling in patients with diabetes, both numerically and by inappropriate orchestration of their microenvironment.

Bone Morphogenetic Protein 6↗
PubMed1998

Bone morphogenetic protein 6 in skeletal metastases from prostate cancer and other common human malignancies.

Prostatic adenocarcinoma commonly metastasizes to bone. Unlike most other bony secondaries, the majority of skeletal prostatic metastases are osteoblastic rather than osteolytic in nature. Several growth factors which are known to stimulate bone formation are expressed in benign and malignant prostate cells, but none has been specifically linked to osteosclerotic metastases. Bone morphogenetic proteins (BMPs) induce ectopic bone formation in vivo. We have reported previously that BMP-6 mRNA and protein are expressed in the majority of primary prostatic carcinomas with established skeletal metastases but rarely in clinically organ-confined tumours. This study examines the expression of BMP-6 mRNA in matched prostatic primary and secondary bony lesions and in isolated skeletal metastases from prostatic adenocarcinomas, as well as other common human malignancies, by in situ hybridization. BMP-6 mRNA was detected in 11 out of 13 bone metastases from prostate carcinoma and in three paired samples of primary prostate carcinoma and matching skeletal metastasis. Weak signals for BMP-6 were also present in 5 out of 17 skeletal deposits from non-prostatic malignancies. BMP-6 mRNA appears to be strongly expressed in prostatic adenocarcinomas, both in the primary tumour and in bone metastases. It is also expressed, though less frequently, in skeletal metastases from other human carcinomas. Our findings suggest that BMP-6 may hold potential as an attractive marker and possible mediator of skeletal metastases, particularly in prostate carcinoma.

Adenocarcinoma↗
PubMed2001

Epigenetic regulation of human bone morphogenetic protein 6 gene expression in prostate cancer.

Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-beta (TGF-beta) superfamily, are multifunctional molecules that regulate bone induction and organ development. Among BMPs, BMP-6 has been shown to be overexpressed in prostate cancer and is speculated to be associated with bone-forming skeletal metastasis. We investigated the regulatory mechanism of the BMP-6 gene expression in prostate cancer cell lines DU-145, LNCaP, PC-3, and PC-3M with regard to the methylation status of the CpG island in the 5' flanking region of the human BMP-6 gene. By sequence-specific analysis of methylated cytosines, we show here that the methylation status of the CpG loci around the Sp1 site of the BMP-6 promoter is related to its steady-state expression and an alternative splicing of messenger RNA (mRNA) in prostate cancer cell lines. Furthermore, a study of clinical cases of benign and malignant prostate lesion by in situ hybridization showed that BMP-6 expression was high at both primary and secondary sites in cases of advanced cancer with metastasis. Demethylation of the CpG loci around the Spl binding site was shown in cases with high BMP-6 expression by sequencing analysis of the methylated cytosine from paraffin-embedded materials. Our results suggested that during cancer progression, besides inactivation of tumor suppressor genes by hypermethylation, activation of certain genes like BMP-6 by selective demethylation was a common epigenetic event giving a variable character to the invading and metastasizing cancer cells.

Aged↗
PubMed1999

Expression, regulation and clinical significance of bone morphogenetic protein 6 in esophageal squamous-cell carcinoma.

Bone morphogenetic proteins (BMPs) are multifunctional cytokines. BMP-6 is involved in early steps of keratinogenesis. Esophageal squamous-cell carcinoma often shows abnormal keratinization. The aim of this study was to examine the expression and regulation of BMP-6 in esophageal carcinoma and to determine the relationship of its expression with tumor characteristics and survival. Avidin-biotin-peroxidase staining was performed using paraffin-wax-embedded tumor specimens collected from 172 patients with esophageal squamous-cell carcinoma, who underwent potentially curative surgery. Expression of BMP-6 and EGF-receptor in the esophageal-squamous-cell-carcinoma cell line COLO-680N and the hypopharyngeal-squamous-cell-carcinoma cell line FaDu was assayed with specific RT-PCR. In standard cell-cultivation conditions, COLO-680N and FaDu cells showed distinct expression of BMP-6, whereas in serum-free conditions BMP-6 expression was hardly detectable. The addition of EGF or EGF-related growth factors could elevate BMP-6 mRNA content. Immunohistochemically, all 172 tumors exhibited cytoplasmic immunoreactivity for BMP-6. The content of BMP-6 protein was inversely correlated with tumor differentiation (p < 0.01). Univariate survival analysis revealed that 74 patients with tumors of low immunoreactivity had higher 2-year and 5-year survival rates (50.0%/27.7%) than 75 patients with tumors of high immunoreactive scores (33.3%/21.6%). (p < 0.05). We conclude that BMP-6 is expressed in esophageal squamous-cell carcinoma. EGF-receptor activation is crucial for regulation of BMP-6 expression. BMP-6 protein content is correlated with the grade of tumor-cell differentiation and may add to indications of poor prognosis.

Adult↗
PubMed2003

Expression of bone morphogenetic protein 6 in healthy and osteoarthritic human articular chondrocytes and stimulation of matrix synthesis in vitro.

OBJECTIVE: To elucidate the role of bone morphogenetic protein 6 (BMP-6) in human articular cartilage, we investigated whether BMP-6 is expressed in adult human articular chondrocytes and analyzed the potential stimulatory effects of BMP-6 on these cells. In addition, we investigated whether osteoarthritic (OA) and normal cartilage chondrocytes behave differently. METHODS: Endogenous expression of the BMP-6 gene was examined by reverse transcription-polymerase chain reaction. BMP-6 protein was detected by Western immunoblotting. Chondrocytes were grown as monolayer cultures for 7 days in a chemically defined serum-free medium, in the absence or presence of recombinant BMP-6. Proteoglycan (PG) synthesis was measured by (35)S-sulfate incorporation into newly synthesized macromolecules. Cell proliferation was assessed by (3)H-thymidine incorporation. RESULTS: BMP-6 was expressed in both healthy and OA chondrocytes at the messenger RNA and protein levels. Total PG synthesis was significantly increased after BMP-6 stimulation of healthy (mean +/- SEM 191 +/- 11%; P < 0.001) and OA (150 +/- 25%; P < 0.03) chondrocyte cultures. A direct comparison between healthy and OA samples revealed no significant difference. The proliferation rates of normal and OA chondrocytes were not affected by BMP-6 treatment. CONCLUSION: BMP-6 is endogenously expressed in chondrocytes obtained from OA and normal adult human articular cartilage. Furthermore, BMP-6 has the potential to stimulate total PG synthesis in human articular chondrocytes derived from normal as well as OA joints. We conclude that the presence of BMP-6 in adult human articular cartilage indicates a functional role for this growth factor in the maintenance of joint integrity.

Adult↗
PubMed1997

Expression of indian hedgehog, bone morphogenetic protein 6 and gli during skeletal morphogenesis.

A complex signaling pathway involving members of the Hedgehog, Bone morphogenetic protein (Bmp) and Gli families regulates early patterning events in fetal skeletogenesis (Hui and Joyner, 1993. A mouse model of Greig cephalopolysyndactyly syndrome: the extra-toes mutation contains an intragenic deletion of the Gli3 gene. Nat. Genet. 3, 241-246; Bitgood and McMahon, 1995. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev. Biol. 172, 126-138; Lanske et al., 1996. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273, 663-666; Vortkamp et al., 1996. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273, 613-622). Hedgehog genes encode secreted proteins that mediate patterning and growth through the induction of secondary signals (reviewed in Hammerschmidt et al., 1997. The world according to hedgehog. Trends Genet. 13, 14-21). Two potential targets of Ihh are bmp6 and gli (Johnson et al., 1995. Patched overexpression alters wing disc size and pattern: transcriptional and post-transcriptional effects on hedgehog targets. Development 121, 4161-4170; Dominguez et al., 1996. Sending and receiving the hedgehog signal: control by the Drosophila Gli protein Cubitus interruptus. Science 272, 1621-1625; Marigo et al., 1996. Sonic hedgehog differentially regulates expression of GLI and GLI3 during limb development. Dev. Biol. 180, 273-283). We investigated the molecular similarities and differences between fetal and postnatal skeletal development by analyzing the coincident and complimentary expression domains of indian hedgehog (ihh), bmp6 and gli in adjacent sections throughout the process of skeletogenesis. In almost all of the skeletal tissues examined, the expression domains of ihh and bmp6 were adjacent to one another and this region was surrounded by gli-expressing cells. These observations are in keeping with the proposed function of gli as a negative regulator of Ihh signaling and the induction of Bmps by Hedgehog proteins (Roberts et al., 1995. Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut. Development 121, 3163-3174; Kawakami et al., 1996. BMP signaling during bone pattern determination in the developing limb. Development 122, 3557-3566). By puberty, ihh, bmp6 and gli transcripts were no longer detected in the growth plate, despite the fact that physeal chondrocytes continued to hypertrophy and differentiate. Although bmp6 was expressed, ihh transcripts were not found in primordia of intramembranous bones, nor in cells lining the future articular surfaces. Collectively our findings suggest that ihh participates in, but is not required for chondrocyte hypertrophy.

Animals↗
PubMedDate not supplied

Human mucosal epithelium involvement in prenatal growth of maxillary sinuses.

The mechanism of formation of the maxillary sinuses is not elucidated as yet, although their morphology during embryogenesis is well described. In the prenatal period, the pneumatization hypothesis is not valid. As the molecular approach to this problem is difficult to apply to human samples, we decided to apply immunohistochemical reactions to analyse the synthesis of selected molecules involved in the rebuilding of tissues. Hematoxylin-eosin staining and immunohistochemical reactions for the detection of MMPs (matrix metalloproteinases), one of their inhibitor TIMP 1 (tissue inhibitor of MMPs), BMP 6 (bone morphogenetic protein 6) and TGF-beta (transforming growth factor beta) were performed in the epithelium the mucosa of the maxillary sinuses of several human foetuses from the collection of the Anatomical Institute. The age of the foetuses was 8, 11, 15, 16, 17, 18 and 22 weeks. An intense positive reaction for MMPs 1, 2 and 3 was found in the mucosal epithehum of developing sinuses in the whole series of foetuses was found. The reaction was more intense in advanced stages of foetal development. Tissue derived inhibitor TIMP was hardly detectable, regardless of the age of samples. However, the intensity of the reaction for TGFbeta was strong in both young and more mature sinus epithelium. The presence of BMP 6, a member of the superfamily of TGFbeta, was detected although the intensity of this reaction in the epithelium was rather weak. Both TGFbeta and BMP 6 are well known as regulators of differentiation in the course of organogenesis. Results of the histochemical analysis suggest the possible involvement of the epithelium in the growth and formation of the maxillary sinuses. The main argument for this is intense reaction for MMP proteases which, as in bone, regulate the turnover and rebuilding processes of the extracellular matrix (ECM).

Body Patterning↗
PubMed1999

Expression of bone morphogenetic protein 6 in normal mammary tissue and breast cancer cell lines and its regulation by epidermal growth factor.

Bone morphogenetic proteins (BMPs) are multifunctional regulators of proliferation, differentiation and apoptosis. BMP-6 is involved in numerous developmental processes. We have demonstrated expression of BMP-6 in breast cancer cell lines by RT-PCR and immuno-histochemistry. The level of BMP-6 mRNA decreased upon serum starvation, whereas epidermal growth factor (EGF) treatment led to elevation of BMP-6 mRNA levels in a dose-dependent manner, with a maximum at 50 ng/ml EGF under serum-free conditions in hormone-sensitive (MCF-7) and in hormone-insensitive (SK-BR-3) breast cancer cell lines. The EGF-like growth factors transforming growth factor-alpha, amphiregulin and betacellulin were also able to elevate the BMP-6 mRNA level after 24 hr. Inhibition of EGF receptor tyrosine kinase with tyrphostine AG 1517 repressed the inductive effect of these growth factors, indicating an EGF receptor-mediated regulation of BMP-6 mRNA. In addition, BMP-6 mRNA was detected in tumor samples from breast carcinoma patients. However, levels were reduced in 18/44 samples compared with tumor-free resection margins. In 12 of these 18 patients, at least a 10-fold reduction of EGF receptor mRNA levels in tumor samples vs. tumor-free samples was observed. This suggests a putative relationship between EGF receptor and BMP-6 mRNA levels in breast cancer.

Bone Morphogenetic Protein 6↗
PubMed2006

Potent induction of chondrocytic differentiation of human adipose-derived adult stem cells by bone morphogenetic protein 6.

OBJECTIVE: Recent studies have identified an abundant source of multipotent progenitor cells in subcutaneous human adipose tissue, termed human adipose-derived adult stem cells (ADAS cells). In response to specific media formulations, including transforming growth factor beta1 (TGFbeta1), these cells exhibit significant ability to differentiate into a chondrocyte-like phenotype, expressing cartilage-specific genes and proteins such as aggrecan and type II collagen. However, the influence of other growth factors on the chondrogenic differentiation of ADAS cells is not fully understood. This study was undertaken to investigate the effects of TGFbeta1, TGFbeta3, insulin-like growth factor 1, bone morphogenetic protein 6 (BMP-6), and dexamethasone, in various combinations, on the chondrogenic potential of ADAS cells in alginate beads. METHODS: The chondrogenic response of alginate-encapsulated ADAS cells was measured by quantitative polymerase chain reaction, 3H-proline and 35S-sulfate incorporation, and immunolabeling for specific extracellular matrix components. RESULTS: Significant differences in chondrogenesis were observed under the different culture conditions for all outcomes measured. Most notably, BMP-6 up-regulated AGC1 and COL2A1 expression by an average of 205-fold and 38-fold, respectively, over day-0 controls, while down-regulating COL10A1 expression by approximately 2-fold. CONCLUSION: These findings suggest that BMP-6 is a potent inducer of chondrogenesis in ADAS cells, in contrast to mesenchymal stem cells, which exhibit increased expression of type X collagen and a hypertrophic phenotype in response to BMP-6. Combinations of growth factors containing BMP-6 may provide a novel means of regulating the differentiation of ADAS cells for applications in the tissue-engineered repair or regeneration of articular cartilage.

Adipose Tissue↗
PubMed1994

Stage-specific expression of decapentaplegic-Vg-related genes 2, 4, and 6 (bone morphogenetic proteins 2, 4, and 6) during human tooth morphogenesis.

Members of the decapentaplegic-Vg-related (DVR) gene family are diffusible signaling molecules regulating inductive tissue interactions during vertebrate development. Expression of DVR/bone morphogenetic protein (BMP) 2, 4, and 6 was studied in human fetal teeth. Sequential morphogenetic stage-specific studies of DVR/BMP 2 and 4 mRNA expression by in situ hybridization revealed transcripts for DVR/BMP 4 during compaction of the dental mesenchyme. In contrast, DVR/BMP 2 mRNA appeared later during tooth development and was located in differentiated cells (odontoblasts). These results were confirmed by reverse-transcription polymerase chain reaction (RT-PCR), which detected DVR/BMP 2 and 4 mRNA in human tooth-germ samples. DVR/BMP 6 protein was distributed in the early dental epithelium and, later, in pre-odontoblasts and odontoblasts, where it remained during dentin formation. These results suggest that DVR/BMP 4 is involved in the early tooth morphogenesis. DVR/BMP 6 may, in particular, be implicated in epithelial-mesenchymal interactions controlling cytodifferentiation. DVR/BMP 2 and 6 may also be involved in odontoblast secretory function. The results suggest that members of the DVR gene family may play regulatory roles during human tooth development.

Base Sequence↗
PubMed2001

Ca2+ and BMP-6 signaling regulate E2F during epidermal keratinocyte differentiation.

The epidermis consists of a squamous epithelium continuously replenished by committed stem cells, which can either self-renew or differentiate. We demonstrated previously that E2F genes are differentially expressed in developing epidermis (Dagnino, L., Fry, C. J., Bartley, S. M., Farnham, P., Gallie, B. L., and Phillips, R. A. (1997) Cell Growth Differ. 8, 553-563). Thus, we hypothesized that various E2F proteins likely play distinct growth regulatory roles in the undifferentiated stem cells and in terminally differentiated keratinocytes. To further understand the function of E2F genes in epidermal morphogenesis, we have examined the expression, regulation, and protein-protein interactions of E2F factors in undifferentiated cultured murine primary keratinocytes or in cells induced to differentiate with Ca(2+) or BMP-6 (bone morphogenetic protein 6). We find similar patterns of E2F regulation with both differentiating agents and demonstrate a switch in expression from E2F-1, -2, and -3 in undifferentiated, proliferating cells to E2F-5 in terminally differentiated keratinocytes. Inhibition of keratinocyte proliferation by transforming growth factor-beta1 did not enhance E2F-5 protein levels, suggesting that this response is specific to differentiation rather than reversible cell cycle withdrawal. E2F-5 up-regulation is also accompanied by formation of heteromeric nuclear complexes containing E2F5, p130, and histone deacetylase (HDAC) 1. Overexpression of E2F5 specifically inhibited DNA synthesis in undifferentiated keratinocytes in an HDAC-dependent manner, suggesting that E2F-5.p130.HDAC1 complexes are likely involved in the permanent withdrawal from the cell cycle of keratinocytes responding to differentiation stimuli.

Animals↗
PubMed2006

Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation.

Oocytes regulate follicle growth by secreting paracrine growth factors that act on neighbouring granulosa cells (GCs). Those factors identified to date are mainly members of the transforming growth factor-beta (TGFbeta) superfamily, but little is known about which specific receptor/signalling system(s) they employ. This study was conducted to determine the requisite pathways utilised by oocytes to promote GC proliferation. We used an established oocyte-secreted mitogen bioassay, where denuded mouse oocytes are co-cultured with mural GCs. Oocytes, growth differentiation factor-9 (GDF9), TGFbeta1 and activin-A all promoted GC DNA synthesis, but bone-morphogenetic protein 6 (BMP6) did not. Subsequently, we tested the capacity of various TGFbeta superfamily receptor ectodomains (ECD) to neutralise oocyte- or specific growth factor-stimulated GC proliferation. The BMP type-II receptor (BMPR-II) ECD antagonised oocyte and GDF9 bioactivity dose-dependently, but had no or minimal effect on TGFbeta1 and activin-A bioactivity, demonstrating its specificity. The TGFbetaR-II, activinR-IIA and activinR-IIB ECDs all failed to neutralise oocyte- or GDF9-stimulated GC DNA synthesis, whereas they did antagonise the activity of their respective native ligands. An activin receptor-like kinase (ALK) 4/5/7 inhibitor, SB431542, also antagonised both oocyte and GDF9 bioactivity in a dose-dependent manner. Consistent with these findings, oocytes, GDF9 and TGFbeta1 all activated SMAD2/3 reporter constructs in transfected GC, and led to phosphorylation of SMAD2 proteins in treated cells. Surprisingly, oocytes did not activate the SMAD1/5/8 pathway in transfected GCs although exogenous BMP6 did. This study indicates that oocyte paracrine factors primarily utilise a similar signalling pathway first identified for GDF9 that employs an unusual combination of TGFbeta superfamily receptors, the BMPR-II and a SMAD2/3 stimulatory ALK (4, 5 or 7), for transmitting their mitogenic actions in GC. This cell-signalling pathway may also have relevance in the hypothalamic-pituitary axis and in germ-somatic cell interactions in the testis.

Activins↗
PubMed2006

The role of bone morphogenetic proteins 2, 4, 6 and 7 during ovarian follicular development in sheep: contrast to rat.

The intraovarian roles of BMP family members such as BMP2, 4, 6 and 7 are not well understood, particularly in species with low ovulation rates such as sheep. Therefore, the objectives of these experiments were to determine the expression patterns of mRNAs encoding BMP2, 4, 6 and 7 during ovarian follicular development in sheep, and to determine the effects of these growth factors on ovine granulosa cell functions in vitro. For comparative purposes, the effects of these BMPs were also determined in rat granulosa cells since these factors have been most widely studied in this poly-ovulatory species. As assessed by in situ hybridization, non-atretic ovine follicles expressed mRNA for BMP6 but not 2, 4 or 7. Furthermore, expression of BMP6 was limited to the oocyte of primordial as well as primary, pre-antral and antral follicles. Reverse transcription-PCR of granulosa cell mRNA detected low levels of all the BMPs in some pools of cells. BMP2, 4, 6 and 7 each inhibited progesterone production from ovine granulosa cells without affecting cellular proliferation/survival. Similarly, these BMPs inhibited progesterone production from rat granulosa cells. However, they also stimulated cellular proliferation/survival of the rat granulosa cells highlighting a species-specific difference for these growth factors. In conclusion, in sheep, BMP2, 4, 6 and 7 inhibit granulosa cell differentiation without affecting proliferation. However, as BMP2, 4 and 7 were not detectable by in situ hybridization in any cells of non-atretic ovarian follicles, it seems unlikely that these proteins would have an important intra-ovarian role in regulating follicular development in sheep. In contrast, localization of BMP6 mRNA in the oocyte suggests that this BMP family member may have a paracrine and/or autocrine role in regulating follicular growth in sheep, as has been shown for two other oocyte derived from members of the transforming growth factor superfamily, BMP15 and growth differentiation factor 9.

Animals↗
PubMed2003

Transcriptional regulation of a BMP-6 promoter by estrogen receptor alpha.

UNLABELLED: The effects of 17beta-estradiol (E2) and ICI 182,780 (ICI) on activity of a BMP-6 promoter were compared in osteoblast-like and breast cancer cells transiently transfected with ERalpha. E2 but not ICI stimulated BMP-6 reporter activity in breast cancer cells, whereas the opposite was observed in osteoblast-like cells, associated with lack of AF-2 dependence of the response, and absent intranuclear localization of ERalpha, suggesting the involvement of a distinct ERalpha-dependent response mechanism in osteoblasts. INTRODUCTION: Previous studies suggest that the tissue-selective effect of antiestrogens on bone reflects the ability of these compounds to target certain osteoblast regulatory genes. To explore this hypothesis, we examined whether antiestrogens preferentially stimulate the bone morphogenetic protein 6 (BMP-6) promoter in bone cells, and if so, whether this activity is associated with a distinct estrogen receptor (ER)alpha-dependent response mechanism to that in other cell types. MATERIALS AND METHODS: We compared the effects of 17beta-estradiol (E2) and ICI 182,780 (ICI) on activity of a 4.3-kb BMP-6 reporter construct in osteoblast-like cells (human MG63 and SaOS-2 cells and rat ROS 17/2.8 cells), human MCF-7 and T47-D breast cancer cell lines, and HepG2 hepatoma cells, after transient transfection with ERalpha, ERbeta, and mutant ER constructs. RESULTS: E2, but not ICI, stimulated BMP-6 reporter activity by approximately 100% in MCF-7, T47-D cells, and HepG2 cells when transfected with ERalpha. In contrast, in ERalpha-transfected osteoblast-like cells, an increase in reporter activity of approximately 75% was observed after treatment with ICI but not E2. The response of MG63 cells to ICI and MCF-7 cells to E2 both required ERalpha as opposed to ERbeta and the ERalpha activation function (AF)-1 activation domain. However, whereas the AF-2 domain was also required for E2 to stimulate reporter activity in MCF-7 cells, the response to ICI in MG63 cells was AF-2 independent. In further studies where we compared the intracellular distribution of ERalpha associated with these responses, E2-dependent stimulation of the BMP-6 reporter in MCF-7 cells was associated with intranuclear localization of ERalpha, whereas extranuclear localization was seen in rat osteosarcoma cells (ROS) cells treated with ICI. CONCLUSIONS: Antiestrogens selectively stimulate BMP-6 reporter activity in osteoblast-like cells through a distinct ERalpha-dependent mechanism characterized by independence of the AF-2 domain and extranuclear localization of ERalpha.

Animals↗
PubMed2004

Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library.

Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, DeltaHexA-GlcNSO(3)-(HexA-GlcNSO(3))(3)) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin. 2-O- and 6-O-sulfated Octa-I were prepared by enzymatically transferring one to three 2-O-sulfate groups and one to three 6-O-sulfate groups per molecule, respectively, to Octa-I. Another octasaccharide containing 3 units of HexA(2SO(4))-GlcNSO(3)(6SO(4)) was prepared also from heparin. This octasaccharide library was subjected to affinity chromatography for interactions with fibroblast growth factor (FGF)-2, -4, -7, -8, -10, and -18, hepatocyte growth factor, bone morphogenetic protein 6, and vascular endothelial growth factor, respectively. Based upon differences in the affinity to those octasaccharides, the growth factors could be classified roughly into five groups: group 1 needed 2-O-sulfate but not 6-O-sulfate (FGF-2); group 2 needed 6-O-sulfate but not 2-O-sulfate (FGF-10); group 3 had the affinity to both 2-O-sulfate and 6-O-sulfate but preferred 2-O-sulfate (FGF-18, hepatocyte growth factor); group 4 required both 2-O-sulfate and 6-O-sulfate (FGF-4, FGF-7); and group 5 hardly bound to any octasaccharides (FGF-8, bone morphogenetic protein 6, and vascular endothelial growth factor). The approach using the oligosaccharide library may be useful to define specific structures required for binding to various heparin-binding proteins. Octasaccharides with the high affinity to FGF-2 and FGF-10 had the activity to release them, respectively, from their complexes with HS. Thus, the library may provide new reagents to specifically regulate bindings of the growth factors to HS.

Amino Acid Sequence↗
PubMed2006

Bone morphogenetic proteins 5 and 6 stimulate osteoclast generation.

Bone regeneration is required for fracture-healing, and different procedures have been used to promote osteogenesis. Recently, BMP-2 has been shown to induce bone formation in vivo and has been tested in clinical trials. A recent in vitro study evaluated the osteogenic activity of 14 BMPs on osteoblastic progenitor cells with an osteogenic hierarchical model in which BMP-2 and BMP-6 may play an important role in inducing osteoblast differentiation. Although the relative osteoinductive activity of each BMP is important, bone regeneration is a process consisting of bone formation and bone resorption. Therefore, it remains unclear which effects BMP-5 and -6 have on the generation of osteoclasts and by which mechanism osteoclastogenesis is stimulated. To compare osteoclastic potency of each BMP, primary murine bone marrow cells were treated with human recombinant BMP-2, BMP-5, or BMP-6 and 1,25-(OH)2 vitamin D3 and stained for the TRAP enzyme. Osteogenic activity of BMP-5 was determined by measuring induction of ALP-activity and proliferation after incubation with primary murine osteoblasts. For elucidating the molecular mechanism, primary bone marrow cells with various concentrations of OPG were added to the TRAP assay and mRNA levels of RANKL and OPG were measured after stimulation with BMP-5. The presented data show that BMP-5 and BMP-6, unlike BMP-2, enhanced the formation of murine TRAP+/MNCs in a biphasic curve. BMP-5 and -6 were less potent in stimulating osteoclastogenesis compared to BMP-2. Concerning the effects of BMP-5 on osteoblasts, there was a dose-dependent increase of ALP activity and proliferation up to a maximum dose of 300 ng/mL. At the mRNA level, BMP-5 increased the RANKL/OPG ratio. In conclusion, this study demonstrates that in contrast to BMP-2, BMP-5 and -6 influences the generation of osteoclasts in a biphasic mode. Both proteins might be very important regulators of bone homeostasis, and therefore, potent candidates for future treatment strategies of bone regeneration.

Acid Phosphatase↗