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FK506 Induces Ligand-Independent Activation of the Bone Morphogenetic Protein Pathway and Osteogenesis.

ABSTRACT: Osteoinductive bone morphogenetic proteins (BMPs), including BMP-2, have a unique capability of mediating bone formation both in orthotopic and ectopic locations. Immunosuppresive macrolides have been shown to potentiate BMP-2 activity through FKBP12, but these have yet to translate to effective osteoinductive therapies. Herein, we show the osteogenic activity of FK506 as a stand-alone agent in direct comparison to BMP-2 both in vitro and in vivo. FK506 was capable of producing stand-alone alkaline phosphatase induction in C2C12 cells comparable to that seen with rhBMP-2. FK506 treatment activated the BMP receptor, as shown by increased pSmad1/5 levels, and produced significantly higher mRNA levels of the early response genes in BMP and TGF-? pathways. Additionally, the FK506 induction of alkaline phosphatase was shown to be resistant to Noggin treatment. In vivo osteogenic activity of FK506 was tested by local delivery on a collagen sponge in an ectopic subcutaneous implantation model in the rat. Dose responses of FK506 showed increasing levels of ectopic mineralization comparable to the mineral volume produced by BMP-2 delivery. These findings suggest that the use of FK506 can enhance osteoblastic differentiation in vitro and can induce mineralization when delivered locally in vivo.

SUBMITTER: Sangadala S

PROVIDER: S-EPMC6515024 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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FK506 Induces Ligand-Independent Activation of the Bone Morphogenetic Protein Pathway and Osteogenesis.

Sangadala Sreedhara S Devereaux Emily J EJ Presciutti Steven M SM Boden Scott D SD Willet Nick J NJ

International journal of molecular sciences 20190417 8

Osteoinductive bone morphogenetic proteins (BMPs), including BMP-2, have a unique capability of mediating bone formation both in orthotopic and ectopic locations. Immunosuppresive macrolides have been shown to potentiate BMP-2 activity through FKBP12, but these have yet to translate to effective osteoinductive therapies. Herein, we show the osteogenic activity of FK506 as a stand-alone agent in direct comparison to BMP-2 both in vitro and in vivo. FK506 was capable of producing stand-alone alkal ...[more]

PMID: 30999619

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Project description:BackgroundProstate cancer is a common cancer in men that annually results in more than 33 000 US deaths. Mortality from prostate cancer is largely from metastatic disease, reflecting on the great strides in the last century of treatments in care for the localized disease. Metastatic castrate resistant prostate cancer (mCRPC) will commonly travel to the bone, creating unique bone pathology that requires nuanced treatments in those sites with surgical, radio and chemotherapeutic interventions. The bone morphogenetic protein (BMP) pathway has been historically studied in the capacity to regulate the osteogenic nature of new bone. New mineralized bone generation is a frequent and common observation in mCRPC and referred to as blastic bone lesions. Less common are bone destructive lesions that are termed lytic.MethodsWe queried the cancer genome atlas (TCGA) prostate cancer databases for the expression of the BMP pathway and found that distinct gene expression of the ligands, soluble antagonists, receptors, and intracellular mediators were altered in localized versus metastatic disease. Human prostate cancer cell lines have an innate ability to promote blastic- or lytic-like bone lesions and we hypothesized that inhibiting BMP signaling in these cell lines would result in a distinct change in osteogenesis gene expression with BMP inhibition.ResultsWe found unique and common changes by comparing these cell lines response and unique BMP pathway alterations. We treated human PCa cell lines with distinct bone pathologic phenotypes with the BMP inhibitor DMH1 and found distinct osteogenesis responses. We analyzed distinct sites of metastatic PCa in the TCGA and found that BMP signaling was selectively altered in commons sites such as lymph node, bone and liver compared to primary tumors.ConclusionsOverall we conclude that BMPs in metastatic prostate cancer are important signals and functional mediators of diverse processes that have potential for individualized precision oncology in mCRPC.

Project description:The use of growth factors for the regeneration of soft and hard tissues has been utilized extensively in dental medicine over the past decade. Recently our group found that recombinant human bone morphogenetic protein 9 (rhBMP9) was more osteopromotive than recombinant human bone morphogenetic protein 2 (rhBMP2) when combined with a deprotenized bovine bone mineral bone grafting material. The aim of the present in vitro study was to evaluate the regenerative potential of an absorbable collagen sponge(ACS) specifically designed for extraction socket healing loaded with rhBMP9 when compared to rhBMP2. The adsorption and release kinetics of rhBMP2 and rhBMP9 were first investigated by enzyme-linked immunosorbent assay quantification. Then, the cellular effects of stromal cell line (ST2) preosteoblasts were investigated utilizing four groups including rhBMP2 and rhBMP9 at both low(10 ng/ml) and high(100 ng/ml) concentrations loaded onto ACS. Cellular attachment(8 hours) and proliferation(1, 3, and 5 days) as well as osteoblast differentiation were investigated by real-time polymerase chain reaction (PCR) at 3 and 14 days, alkaline phosphatase (ALP) activity at 7 days, and alizarin red staining at 14 days. ACS fully adsorbed both rhBMP2 and rhBMP9 that were slowly released up to 10 days. Although neither rhBMP2 nor rhBMP9 had any effects on cell attachment or proliferation, pronounced effects were observed on osteoblast differentiation. ALP activity was increased seven-fold with rhBMP2-high, whereas a marked 10-fold and 20-fold increase was observed with rhBMP9-low and high loaded to ACS, respectively. Furthermore, mRNA levels of collagen1, ALP, bone sialoprotein, and osteocalcin were all significantly higher for rhBMP9 when compared to control or rhBMP2 groups. Alizarin red staining further confirmed that rhBMP9-low and high demonstrated marked increases in mineralization potential when compared to rhBMP2-high. The results demonstrate the marked effect of rhBMP9 on osteoblast differentiation when combined with ACS in comparison to rhBMP2 at doses as much as 10 times lower. Further in vivo studies are necessary to investigate whether the regenerative potential is equally as potent.

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FK506 Induces Ligand-Independent Activation of the Bone Morphogenetic Protein Pathway and Osteogenesis.

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FK506 Induces Ligand-Independent Activation of the Bone Morphogenetic Protein Pathway and Osteogenesis.

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