Inhibition of Transforming Growth Factor β Activation Diminishes Tumor Progression and Osteolytic Bone Disease in Mouse Models of Multiple Myeloma

Authors

Ailing Lu, Manuel Antonio Pallero, Weiqi Lei, Huixian Hong, Yang Yang, Mark J. Suto, Joanne E. Murphy-Ullrich

Abstract

Transforming growth factor (TGF)-β supports multiple myeloma progression and associated osteolytic bone disease. Conversion of latent TGF-β to its biologically active form is a major regulatory node in controlling its activity. Thrombospondin1 (TSP1) binds and activates TGF-β. TSP1 is increased in myeloma; TSP1–TGF-β activation inhibits osteoblast differentiation. We hypothesized that TSP1 regulates TGF-β activity in myeloma and antagonizing TSP1–TGF-β axis may inhibit myeloma progression. Antagonists (LSKL peptide, SRI31277) derived from the LSKL sequence of latent TGF-β that block TSP1–TGF-β activation were used to determine the role of the TSP1–TGF-β pathway in three mouse models of myeloma. TSP1 bound to human myeloma cells and activated latent TGF-β produced by human and mouse myeloma cell lines. Treatment with antagonists delivered via osmotic pump in an intratibial severe combined immunodeficiency CAG myeloma model or in a systemic severe combined immunodeficiency CAG-heparanase model of aggressive myeloma reduced tumor burden, mouse interleukin-6, and osteoclasts, increased osteoblast number, and inhibited bone destruction as measured by microcomputed tomography. Antagonists reduced TGF-β signaling (phospho-Smad2) in bone sections and reduced tumor burden in the immune competent 5TGM1 model of mouse myeloma. SRI31277 was as effective as dexamethasone or bortezomib, and SRI31277 combined with bortezomib showed greater tumor reduction than either agent alone. These studies validate TSP1-regulated TGF-β activation as a therapeutic strategy for targeted inhibition of TGF-β in myeloma.

Link to Article

http://dx.doi.org/10.1016/j.ajpath.2015.11.003

Growth and repair factors, osteoactivin, matrix metalloproteinase and heat shock protein 72, increase with resolution of inflammation in musculotendinous tissues in a rat model of repetitive grasping

Authors

Nagat Frara, Samir M. Abdelmagid, Michael Tytell, Mamta Amin, Steven N. Popoff, Fayez F. Safadi and Mary F. Barbe

Abstract

Expression of the growth factor osteoactivin (OA) increases during tissue degeneration and regeneration, fracture repair and after denervation-induced disuse atrophy, concomitant with increased matrix metalloproteinases (MMPs). However, OA’s expression with repetitive overuse injuries is unknown. The aim of this study was to evaluate: 1) OA expression in an operant rat model of repetitive overuse; 2) expression of MMPs; 3) inflammatory cytokines indicative of injury or inflammation; and 4) the inducible form of heat shock protein 70 (HSPA1A/HSP72) as the latter is known to increase during metabolic stress and to be involved in cellular repair. Young adult female rats performed a high repetition negligible force (HRNF) food retrieval task for up to 6 weeks and were compared to control rats.

Flexor digitorum muscles and tendons were collected from 22 young adult female rats performing a HRNF reaching task for 3 to 6 weeks, and 12 food restricted control (FRC) rats. OA mRNA levels were assessed by quantitative polymerase chain reaction (qPCR). OA, MMP-1, -2, -3, and -13 and HSP72 protein expression was assayed using Western blotting. Immunohistochemistry and image analysis was used to evaluate OA and HSP72 expression. ELISA was performed for HSP72 and inflammatory cytokines.

Flexor digitorum muscles and tendons from 6-week HRNF rats showed increased OA mRNA and protein expression compared to FRC rats. MMP-1, -2 and -3 progressively increased in muscles whereas MMP-1 and -3 increased in tendons with HRNF task performance. HSP72 increased in 6-week HRNF muscles and tendons, compared to controls, and co-localized with OA in the myofiber sarcolemma. IL-1alpha and beta increased transiently in tendons or muscles in HRNF week 3 before resolving in week 6.

The simultaneous increases of OA with factors involved in tissue repair (MMPs and HSP72) supports a role of OA in tissue regeneration after repetitive overuse.

Link to Article

http://dx.doi.org/10.1186/s12891-016-0892-3

Enamel Matrix Derivative Promotes Healing of a Surgical Wound in the Rat Oral Mucosa

Authors

Tal Maymon-Gil, Evgeny Weinberg, Carlos Nemcovsky, Miron Weinreb

Abstract

Background: Enamel Matrix Proteins (EMPs) play a role in enamel formation and the development of the periodontium. Sporadic clinical observations of periodontal regeneration treatments with Enamel Matrix Derivative (EMD), a commercial formulation of EMPs, suggest it also promotes post-surgical healing of soft tissues. In vitro studies showed that EMD stimulates various cellular effects, which could potentially enhance wound healing. This study examined the in-vivo effects of EMD on healing of an oral mucosa surgical wound in rats.

Methods: A bilateral oral mucosa wound was created via a crestal incision at the anterior edentulous maxilla of Sprague-Dawley rats. Full thickness flaps were raised and following suturing, EMD was injected underneath the soft tissues on one side while the EMD vehicle was injected in the contralateral side. Animals were sacrificed after 5 or 9 days and the wound area was subjected to histological and immunohistochemical analysis of epithelial gap, number of macrophages, blood vessels, proliferating cells and collagen content in the connective tissue. Gene expression analysis was also conducted 2 days after surgery.

Results: EMD had no effect on epithelial gap of the wound. On both days 5 and 9 EMD treatment increased significantly the number of blood vessels and the collagen content. EMD also enhanced (by 20-40%) the expression of transforming growth factor (TGF) β1 and TGFβ2, vascular endothelial growth factor (vEGF), interleukin-1β (IL-1β), matrix metalloproteinase-1 (MMP-1), Versican and Fibronectin.

Conclusions: EMD improves oral mucosa incisional wound healing by promoting formation of blood vessels and collagen fibers in the connective tissue.

Link to Article

http://dx.doi.org/10.1902/jop.2016.150567

Conditional Deletion of Murine Fgf23: Interruption of the Normal Skeletal Responses to Phosphate Challenge and Rescue of Genetic Hypophosphatemia

Authors

Erica L. Clinkenbeard, Taryn A. Cass, Pu Ni, Julia M. Hum, Teresita Bellido, Matthew R. Allen and Kenneth E. White

Abstract

The transgenic and knock out (KO) animals involving Fgf23 have been highly informative in defining novel aspects of mineral metabolism, but are limited by shortened life span, inability of spatial/temporal FGF23 control, and infertility of the global KO. To more finely test the role of systemic and genetic influences in FGF23 production, a mouse was developed that carried a floxed (‘f’)-Fgf23 allele (exon 2 floxed) which demonstrated in vivo recombination when bred to global-Cre transgenic mice (eIIa-cre). Mice homozygous for the recombined allele (‘Δ’) had undetectable serum intact FGF23, elevated serum phosphate (p < 0.05), and increased kidney Cyp27b1 mRNA (p < 0.05) similar to global Fgf23-KO mice. To isolate cellular FGF23 responses during phosphate challenge Fgf23Δ/f mice were mated with early osteoblast type Iα1 collagen 2.3kb promoter-cre mice (Col2.3-cre) and the late osteoblast/early osteocyte Dentin matrix protein-1-cre (Dmp1-cre). Fgf23Δ/f/Col2.3-cre+ and Fgf23Δ/f/Dmp1-cre+ exhibited reduced baseline serum intact FGF23 versus controls. After challenge with high phosphate diet Cre- mice had 2.1-2.5 fold increased serum FGF23 (p < 0.01), but Col2.3-cre+ mice had no significant increase, and Dmp1-cre+ mice had only a 37% increase (p < 0.01) despite prevailing hyperphosphatemia in both models. The Fgf23Δ/f/Col2.3-cre was bred onto the Hyp (murine XLH model) genetic background to test the contribution of osteoblasts and osteocytes to elevated FGF23 and Hyp disease phenotypes. Whereas Hyp mice maintained inappropriately elevated FGF23 considering their marked hypophosphatemia, Hyp/Fgf23Δ/f/Col2.3-cre+ mice had serum FGF23 <4% of Hyp (p < 0.01), and this targeted restriction normalized serum phosphorus and ricketic bone disease. In summary, deleting FGF23 within early osteoblasts and osteocytes demonstrated that both cell types contribute to baseline circulating FGF23 concentrations, and that targeting osteoblasts/osteocytes for FGF23 production can modify systemic responses to changes in serum phosphate concentrations and rescue the Hyp genetic syndrome.

Link to Article

http://dx.doi.org/10.1002/jbmr.2792

Gata2 is a Rheostat for Mesenchymal Stem Cell Fate in Male Mice

Authors

Xiaoxiao Li, HoangDinh Huynh, Hao Zuo, Marjo Salminen, and Yihong Wan

Abstract

Gata2 is a zinc finger transcription factor that is important in hematopoiesis and neuronal development. However, the roles of Gata2 in the mesenchymal lineages are poorly understood. In vitro studies suggest that Gata2 modulates adipocyte differentiation and mesenchymal stem cells (MSC) proliferation. To systematically determine the in vivo functions of Gata2 in MSC lineage commitment and development, we have generated three mouse models where Gata2 is specifically deleted in MSC, adipocyte or osteoblast. During MSC expansion stage, Gata2 promotes proliferation and attenuates differentiation; thereby Gata2 loss in MSC results in enhanced differentiation of both adipocyte and osteoblast. During differentiation stage, Gata2 also plays MSC-independent roles to impede lineage commitment; hence Gata2 loss in adipocyte or osteoblast lineages also augments adipogenesis and osteoblastogenesis, respectively. These findings reveal Gata2 as a crucial rheostat of MSC fate to control osteoblast and adipocyte lineage development.

Link to Article

http://dx.doi.org/10.1210/en.2015-1827

Sclerostin antibody (Scl-Ab) improves osteomalacia phenotype in dentin matrix protein 1(Dmp1) knockout mice with little impact on serum levels of phosphorus and FGF23

Authors

Yinshi Rena, Xianglong Hana, Yan Jinga, Baozhi Yuanc, Huazhu Ked, Min Liud, Jian Q. Feng

Abstract

Unlike treatments for most rickets, the treatment using 1,25-(OH)2 vitamin D3 has little efficacy on patients with hypophosphatemic rickets, a set of rare genetic diseases. Thus, understanding the local cause for osteomalacia in hypophosphatemic rickets and developing an effective treatment to restore mineralization in this rare disease has been a longstanding goal in medicine. Here, we used Dmp1 knockout (KO) mice (whose mutations led to the same type of autosomal recessive hypophosphatemic rickets in humans) as the model in which the monoclonal antibody of sclerostin (Scl-Ab) was tested in two age groups for 8 weeks: the prevention group (starting at age 4 weeks) and the treatment group (starting at age 12 weeks). Applications of Scl-Ab greatly improved the osteomalacia phenotype (> 15%) and the biomechanical properties (3-point bending, ~ 60%) in the treated long-bone group. Our studies not only showed improvement of the osteomalacia in the alveolar bone, which has the highest bone metabolism rate, as well as the long bone phenotypes in treated mice. All these improvements attributed to the use of Scl-Ab are independent of the change in serum levels of phosphorus and FGF23, since Scl-Ab had little efficacy on those parameters. Finally, we propose a model to explain how Scl-Ab can improve the Dmp1 KO osteomalacia phenotype, in which the sclerostin level is already low.

Link to Article

http://dx.doi.org/10.1016/j.matbio.2015.12.009