Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation

Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.

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IGF-binding protein-5 stimulates osteoblast activity and bone accretion in ovariectomized mice

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2001.281.2.e283 ◽

2001 ◽

Vol 281 (2) ◽

pp. E283-E288 ◽

Cited By ~ 33

Author(s):

Dennis L. Andress

Keyword(s):

Bone Formation ◽

Binding Protein ◽

Formation Rate ◽

Bone Matrix ◽

Secretory Protein ◽

Mineral Density ◽

Bone Formation Rate ◽

Ovariectomized Mice ◽

Osteoblast Activity

Insulin-like growth factor binding protein-5 (IGFBP-5) is an osteoblast secretory protein that becomes incorporated into the mineralized bone matrix. In osteoblast cultures, IGFBP-5 stimulates cell proliferation by an IGF-independent mechanism. To evaluate whether IGFBP-5 can stimulate osteoblast activity and enhance bone accretion in a mouse model of osteoblast insufficiency, daily subcutaneous injections of either intact [IGFBP-5 (intact)] or carboxy-truncated IGFBP-5 [IGFBP-5-(1–169)] were given to ovariectomized (OVX) mice for 8 wk. Femur and spine bone mineral density (BMD), measured every 2 wk, showed early and sustained increases in response to IGFBP-5. Bone histomorphometry of cancellous bone showed significant elevations in the bone formation rate in both the femur metaphysis [IGFBP-5- (1)] only) and spine compared with OVX controls. IGFBP-5 also stimulated osteoblast number in the femur IGFBP-5-(1–169) only) and spine. These data indicate that IGFBP-5 effectively enhances bone formation and bone accretion in OVX mice by stimulating osteoblast activity. The finding that IGFBP-5-(1–169) is bioactive in vivo indicates that the carboxy-terminal portion is not required for this bone anabolic effect.

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Nrf Transcription Factors in Keratinocytes Are Essential for Skin Tumor Prevention but Not for Wound Healing

Molecular and Cellular Biology ◽

10.1128/mcb.26.10.3773-3784.2006 ◽

2006 ◽

Vol 26 (10) ◽

pp. 3773-3784 ◽

Cited By ~ 88

Author(s):

Ulrich auf dem Keller ◽

Marcel Huber ◽

Tobias A. Beyer ◽

Angelika Kümin ◽

Christina Siemes ◽

...

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Wound Repair ◽

Target Genes ◽

Dominant Negative ◽

Skin Tumor ◽

Cellular Stress Response ◽

Skin Development

ABSTRACT The Nrf2 transcription factor is a key player in the cellular stress response through its regulation of cytoprotective genes. In this study we determined the role of Nrf2-mediated gene expression in keratinocytes for skin development, wound repair, and skin carcinogenesis. To overcome compensation by the related Nrf1 and Nrf3 proteins, we expressed a dominant-negative Nrf2 mutant (dnNrf2) in the epidermis of transgenic mice. The functionality of the transgene product was verified in vivo using mice doubly transgenic for dnNrf2 and an Nrf2-responsive reporter gene. Surprisingly, no abnormalities of the epidermis were observed in dnNrf2-transgenic mice, and even full-thickness skin wounds healed normally. However, the onset, incidence, and multiplicity of chemically induced skin papillomas were strikingly enhanced, whereas the progression to squamous cell carcinomas was unaltered. We provide evidence that the enhanced tumorigenesis results from reduced basal expression of cytoprotective Nrf target genes, leading to accumulation of oxidative damage and reduced carcinogen detoxification. Our results reveal a crucial role of Nrf-mediated gene expression in keratinocytes in the prevention of skin tumors and suggest that activation of Nrf2 in keratinocytes is a promising strategy to prevent carcinogenesis of this highly exposed organ.

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Osteoblastic Responses to TGF-β during Bone Remodeling

Molecular Biology of the Cell ◽

10.1091/mbc.9.7.1903 ◽

1998 ◽

Vol 9 (7) ◽

pp. 1903-1918 ◽

Cited By ~ 161

Author(s):

Adrian Erlebacher ◽

Ellen H. Filvaroff ◽

Jian-Qin Ye ◽

Rik Derynck

Keyword(s):

Bone Resorption ◽

Bone Formation ◽

Transgenic Mice ◽

Bone Remodeling ◽

Osteoblast Differentiation ◽

Bone Matrix ◽

Physiological Role ◽

Dominant Negative ◽

Central Component ◽

Physiological Regulator

Bone remodeling depends on the spatial and temporal coupling of bone formation by osteoblasts and bone resorption by osteoclasts; however, the molecular basis of these inductive interactions is unknown. We have previously shown that osteoblastic overexpression of TGF-β2 in transgenic mice deregulates bone remodeling and leads to an age-dependent loss of bone mass that resembles high-turnover osteoporosis in humans. This phenotype implicates TGF-β2 as a physiological regulator of bone remodeling and raises the question of how this single secreted factor regulates the functions of osteoblasts and osteoclasts and coordinates their opposing activities in vivo. To gain insight into the physiological role of TGF-β in bone remodeling, we have now characterized the responses of osteoblasts to TGF-β in these transgenic mice. We took advantage of the ability of alendronate to specifically inhibit bone resorption, the lack of osteoclast activity in c-fos −/− mice, and a new transgenic mouse line that expresses a dominant-negative form of the type II TGF-β receptor in osteoblasts. Our results show that TGF-β directly increases the steady-state rate of osteoblastic differentiation from osteoprogenitor cell to terminally differentiated osteocyte and thereby increases the final density of osteocytes embedded within bone matrix. Mice overexpressing TGF-β2 also have increased rates of bone matrix formation; however, this activity does not result from a direct effect of TGF-β on osteoblasts, but is more likely a homeostatic response to the increase in bone resorption caused by TGF-β. Lastly, we find that osteoclastic activity contributes to the TGF-β–induced increase in osteoblast differentiation at sites of bone resorption. These results suggest that TGF-β is a physiological regulator of osteoblast differentiation and acts as a central component of the coupling of bone formation to resorption during bone remodeling.

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Signalling Alterations in Bones of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Gene Deficient Mice

International Journal of Molecular Sciences ◽

10.3390/ijms19092538 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2538 ◽

Cited By ~ 8

Author(s):

Gergő Józsa ◽

Vince Szegeczki ◽

Andrea Pálfi ◽

Tamás Kiss ◽

Zsuzsanna Helyes ◽

...

Keyword(s):

Bone Formation ◽

Adenylate Cyclase ◽

Morphological Changes ◽

Bone Matrix ◽

Transverse Diameter ◽

Type I ◽

Pituitary Adenylate Cyclase ◽

Deficient Mice

: Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with diverse developmental roles, including differentiation of skeletal elements. It is a positive regulatory factor of chondrogenesis and osteogenic differentiation in vitro, but little is known about its in vivo role in bone formation. In our experiments, diaphyses of long bones from hind limbs of PACAP gene-deficient mice showed changes in thickness and increased staining intensity. Our main goal was to perform a detailed morphological and molecular biological analysis of femurs from PACAP knockout (KO) and wild type (WT) mice. Transverse diameter and anterior cortical bone thickness of KO femurs showed significant alterations with disturbed Ca2+ accumulation and collagen type I expression. Higher expression and activity of alkaline phosphatase were also observed, accompanied by increased fragility PACAP KO femurs. Increased expression of the elements of bone morphogenic protein (BMP) and hedgehog signalling was also observed, and are possibly responsible for the compensation mechanism accounting for the slight morphological changes. In summary, our results show that lack of PACAP influences molecular and biomechanical properties of bone matrix, activating various signalling cascade changes in a compensatory fashion. The increased fragility of PACAP KO femur further supports the role of endogenous PACAP in in vivo bone formation.

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The Cocktail Effect of BMP-2 and TGF-β1 Loaded in Visible Light-Cured Glycol Chitosan Hydrogels for the Enhancement of Bone Formation in a Rat Tibial Defect Model

Marine Drugs ◽

10.3390/md16100351 ◽

2018 ◽

Vol 16 (10) ◽

pp. 351 ◽

Cited By ~ 18

Author(s):

Sun-Jung Yoon ◽

Youngbum Yoo ◽

Sang Nam ◽

Hoon Hyun ◽

Deok-Won Lee ◽

...

Keyword(s):

Growth Factor ◽

Bone Formation ◽

Visible Light ◽

Type I ◽

Mineral Density ◽

Glycol Chitosan ◽

Defect Sites ◽

Tgf Β1

Bone tissue engineering scaffolds offer the merits of minimal invasion as well as localized and controlled biomolecule release to targeted sites. In this study, we prepared injectable hydrogel systems based on visible light-cured glycol chitosan (GC) hydrogels containing bone morphogenetic protein-2 (BMP-2) and/or transforming growth factor-beta1 (TGF-β1) as scaffolds for bone formation in vitro and in vivo. The hydrogels were characterized by storage modulus, scanning electron microscopy (SEM) and swelling ratio analyses. The developed hydrogel systems showed controlled releases of growth factors in a sustained manner for 30 days. In vitro and in vivo studies revealed that growth factor-loaded GC hydrogels have no cytotoxicity against MC3T3-E1 osteoblast cell line, improved mRNA expressions of alkaline phosphatase (ALP), type I collagen (COL 1) and osteocalcin (OCN), and increased bone volume (BV) and bone mineral density (BMD) in tibia defect sites. Moreover, GC hydrogel containing BMP-2 (10 ng) and TGF-β1 (10 ng) (GC/BMP-2/TGF-β1-10 ng) showed greater bone formation abilities than that containing BMP-2 (5 ng) and TGF-β1 (5 ng) (GC/BMP-2/TGF-β1-5 ng) in vitro and in vivo. Consequently, the injectable GC/BMP-2/TGF-β1-10 ng hydrogel may have clinical potential for dental or orthopedic applications.

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Pregnancy-Associated Plasma Protein-A Increases Osteoblast Proliferation in Vitro and Bone Formation in Vivo

Endocrinology ◽

10.1210/en.2006-1055 ◽

2006 ◽

Vol 147 (12) ◽

pp. 5653-5661 ◽

Cited By ~ 66

Author(s):

Xuezhong Qin ◽

Jon E. Wergedal ◽

Mark Rehage ◽

Kiet Tran ◽

Jacqueline Newton ◽

...

Keyword(s):

Bone Formation ◽

Transgenic Mice ◽

Plasma Protein ◽

Type I ◽

Osteoblast Proliferation ◽

Bone Formation Rate ◽

Igf I ◽

Proliferation In Vitro

Pregnancy-associated plasma protein (PAPP)-A, a protease for IGF binding protein (IGFBP)-2, -4, and -5, may enhance IGF action by increasing its bioavailability. Here we have determined the role and mechanism of action of PAPP-A in the regulation of osteoblast proliferation in vitro and bone metabolism in vivo. Recombinant PAPP-A (100 ng/ml) significantly increased osteoblast proliferation and free IGF-I concentration. These effects were abolished by noncleavable IGFBP-4, suggesting that PAPP-A promotes osteoblast proliferation by increasing IGF bioavailability. To determine whether PAPP-A exerts an anabolic effect on bone in vivo, we developed transgenic mice that overexpress PAPP-A in osteoblasts using the 2.3-kb rat type I collagen promoter. Consistent with the increase in IGFBP-4 proteolysis, free IGF-I concentration was significantly increased in the conditioned medium of cultured osteoblasts derived from transgenic mice compared with the wild-type littermates. Calvarial bone thickness, bone marrow cavity, and skull bone mineral density were significantly increased in transgenic mice. Bone size-related parameters in femur and tibia such as total bone area and periosteal circumference as determined by peripheral quantitated computed tomography and histological analysis were significantly increased in transgenic mice. Bone formation rate and osteoid surface were increased by more than 2-fold, whereas bone resorbing surface was unaffected. These anabolic effects were sustained with aging. These findings provide strong evidence that PAPP-A acts as a potent anabolic factor in the regulation of bone formation. Thus, enhancing IGF bioavailability by PAPP-A can be a powerful strategy in the treatment of certain metabolic diseases such as osteoporosis.

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A novel gelatin/chitooligosaccharide/demineralized bone matrix composite scaffold and periosteum-derived mesenchymal stem cells for bone tissue engineering

Biomaterials Research ◽

10.1186/s40824-021-00220-y ◽

2021 ◽

Vol 25 (1) ◽

Author(s):

Thakoon Thitiset ◽

Siriporn Damrongsakkul ◽

Supansa Yodmuang ◽

Wilairat Leeanansaksiri ◽

Jirun Apinun ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Bone Tissue Engineering ◽

Bone Tissue ◽

Demineralized Bone Matrix ◽

Bone Matrix ◽

Alp Activity

Abstract Background A novel biodegradable scaffold including gelatin (G), chitooligosaccharide (COS), and demineralized bone matrix (DBM) could play a significant part in bone tissue engineering. The present study aimed to investigate the biological characteristics of composite scaffolds in combination of G, COS, and DBM for in vitro cell culture and in vivo animal bioassays. Methods Three-dimensional scaffolds from the mixture of G, COS, and DBM were fabricated into 3 groups, namely, G, GC, and GCD using a lyophilization technique. The scaffolds were cultured with mesenchymal stem cells (MSCs) for 4 weeks to determine biological responses such as cell attachment and cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, cell morphology, and cell surface elemental composition. For the in vivo bioassay, G, GC, and GCD, acellular scaffolds were implanted subcutaneously in 8-week-old male Wistar rats for 4 weeks and 8 weeks. The explants were assessed for new bone formation using hematoxylin and eosin (H&E) staining and von Kossa staining. Results The MSCs could attach and proliferate on all three groups of scaffolds. Interestingly, the ALP activity of MSCs reached the greatest value on day 7 after cultured on the scaffolds, whereas the calcium assay displayed the highest level of calcium in MSCs on day 28. Furthermore, weight percentages of calcium and phosphorus on the surface of MSCs after cultivation on the GCD scaffolds increased when compared to those on other scaffolds. The scanning electron microscopy images showed that MSCs attached and proliferated on the scaffold surface thoroughly over the cultivation time. Mineral crystal aggregation was evident in GC and greatly in GCD scaffolds. H&E staining illustrated that G, GC, and GCD scaffolds displayed osteoid after 4 weeks of implantation and von Kossa staining confirmed the mineralization at 8 weeks in G, GC, and GCD scaffolds. Conclusion The MSCs cultured in GCD scaffolds revealed greater osteogenic differentiation than those cultured in G and GC scaffolds. Additionally, the G, GC, and GCD scaffolds could promote in vivo ectopic bone formation in rat model. The GCD scaffolds exhibited maximum osteoinductive capability compared with others and may be potentially used for bone regeneration.

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Essential role of IPS-1 in innate immune responses against RNA viruses

Journal of Experimental Medicine ◽

10.1084/jem.20060792 ◽

2006 ◽

Vol 203 (7) ◽

pp. 1795-1803 ◽

Cited By ~ 345

Author(s):

Himanshu Kumar ◽

Taro Kawai ◽

Hiroki Kato ◽

Shintaro Sato ◽

Ken Takahashi ◽

...

Keyword(s):

Rna Viruses ◽

Rna Virus ◽

Critical Role ◽

Nuclear Factor Κb ◽

Type I ◽

Innate Immune Responses ◽

Antiviral Responses ◽

Deficient Mice

IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

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Impaired Alignment of Bone Matrix Microstructure Associated with Disorganized Osteoblast Arrangement in Malignant Melanoma Metastasis

Biomolecules ◽

10.3390/biom11020131 ◽

2021 ◽

Vol 11 (2) ◽

pp. 131

Author(s):

Aira Matsugaki ◽

Yumi Kimura ◽

Ryota Watanabe ◽

Fumihito Nakamura ◽

Ryo Takehana ◽

...

Keyword(s):

Malignant Melanoma ◽

Cell Activation ◽

Ex Vivo ◽

Bone Matrix ◽

Melanoma Metastasis ◽

Mineral Density ◽

High Fracture ◽

Matrix Microstructure ◽

Metastasis Model

Malignant melanoma favors spreading to bone, resulting in a weakened bone with a high fracture risk. Here, we revealed the disorganized alignment of apatite crystals in the bone matrix associated with the homing of cancer cells by developing an artificially controlled ex vivo melanoma bone metastasis model. The ex vivo metastasis model reflects the progressive melanoma cell activation in vivo, resulting in decreased bone mineral density and expression of MMP1-positive cells. Moreover, less organized intercellular connections were observed in the neighboring osteoblasts in metastasized bone, indicating the abnormal and randomized organization of bone matrix secreted by disconnected osteoblasts. Our study revealed that the deteriorated microstructure associated with disorganized osteoblast arrangement was a determinant of malignant melanoma-related bone dysfunction.

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Parathyroid hormone induces bone formation in phosphorylation-deficient PTHR1 knockin mice

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00380.2011 ◽

2012 ◽

Vol 302 (10) ◽

pp. E1183-E1188 ◽

Cited By ~ 6

Author(s):

Nabanita S. Datta ◽

Tareq A. Samra ◽

Abdul B. Abou-Samra

Keyword(s):

Parathyroid Hormone ◽

Bone Formation ◽

Physiological Role ◽

Receptor Internalization ◽

Mineral Density ◽

Trabecular Thickness ◽

Receptor Complexes ◽

Diaphyseal Bone ◽

Anabolic Action

Activation of G protein-coupled receptors by agonists leads to receptor phosphorylation, internalization of ligand receptor complexes, and desensitization of hormonal response. The role of parathyroid hormone (PTH) receptor 1, PTHR1, is well characterized and known to regulate cellular responsiveness in vitro. However, the role of PTHR1 phosphorylation in bone formation is yet to be investigated. We have previously demonstrated that impaired internalization and sustained cAMP stimulation of phosphorylation-deficient (PD) PTHR1 leads to exaggerated cAMP response to subcutaneous PTH infusion in a PD knockin mouse model. To understand the physiological role of receptor internalization on PTH bone anabolic action, we examined bone parameters of wild-type (WT) and PD knockin female and male mice following PTH treatment. We found a decrease in total and diaphyseal bone mineral density in female but not in male PD mice compared with WT controls at 3–6 mo of age. This effect was attenuated at older age groups. PTH administration displayed increased bone volume and trabecular thickness in the vertebrae and distal femora of both WT and PD animals. These results suggest that PTHR1 phosphorylation does not play a major role in the anabolic action of PTH.

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