Publications by authors named "Yuji Mishina"

235 Publications

mTOR signaling regulates gastric epithelial progenitor homeostasis and gastric tumorigenesis via MEK1-ERKs and BMP-Smad1 pathways.

Cell Rep 2021 May;35(5):109069

Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; Center for Traditional Chinese Medicine and Stem Cell Research, the Chengdu University of Traditional Chinese Medicine, Sichuan, China. Electronic address:

mTOR, the sensor of nutrients and growth factors, has important roles in tissue homeostasis and tumorigenesis. However, how mTOR controls gastric epithelial cell turnover and gastric cancer development, a leading malignancy, remains poorly understood. Here, we provide genetic evidence that mTOR activation promotes proliferation and inhibits differentiation of Lgr5 gastric epithelial progenitors (GEPs) in gastric homeostasis and tumorigenesis. mTOR signaling increases MEK1 and Smad1 expression and enhances activation of MEK1-ERKs and BMP-Smad1 pathways, respectively, in GEPs and gastric tumors. Mek1 deletion or inhibition rescues hyperproliferation, whereas Bmpr1a ablation or inhibition rescues differentiation defects of Tsc1 GEPs. Tsc1 deficiency in Lgr5 GEPs accelerates gastric tumor initiation and development, which require MEK1-ERKs for hyperplasia and BMP-Smad1 for differentiation suppression. These findings reveal how mTOR signaling controls Lgr5 GEP homeostasis and cancerization and suggest that ERKs and Smad1 signaling can be safely targeted to substitute mTOR inhibitors in gastric cancer therapy.
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http://dx.doi.org/10.1016/j.celrep.2021.109069DOI Listing
May 2021

Generation of a new mouse line with conditionally activated signaling through the BMP receptor, ACVR1: A tool to characterize pleiotropic roles of BMP functions.

Genesis 2021 Apr 14:e23419. Epub 2021 Apr 14.

Department of Biologic and Materials Sciences & Prosthodontics, School of Dentistry, University of Michigan, MI, USA.

BMP signaling plays pleiotropic roles in various tissues during embryogenesis and after birth. We have previously generated a constitutively activated Acvr1(ca-Acvr1) transgenic mouse line (line L35) through pronuclei injection to investigate impacts of enhanced BMP signaling in a tissue specific manner. However, line L35 shows a restricted expression pattern of the transgene. Here, we generated another ca-Acvr1 transgenic line, line A11, using embryonic stem (ES) transgenesis. The generated line A11 shows distinctive phenotypes from line L35, along with very limited expression levels of the transgene. When the transgene is activated in the neural crest cells in a Cre-dependent manner, line A11 exhibits cleft palate and shorter jaws, while line L35 develops ectopic cartilages and highly hypomorphic facial structures. When activated in limb buds, line A11 develops organized but smaller limb skeletal structures, while line L35 forms disorganized limbs with little mineralization. Additionally, no heterotopic ossification (HO) is identified in line A11 when bred with NFATc1-Cre mice even after induction of tissue injury, which is an established protocol for HO for line L35. Therefore, the newly generated conditional ca-Acvr1 mouse line A11 provides an additional resource to dissect highly context dependent functions of BMP signaling in development and disease.
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http://dx.doi.org/10.1002/dvg.23419DOI Listing
April 2021

Macropore design of tissue engineering scaffolds regulates mesenchymal stem cell differentiation fate.

Biomaterials 2021 May 24;272:120769. Epub 2021 Mar 24.

Department of Biologic and Materials Sciences & Prosthodontics, School of Dentistry, University of Michigan, Ann Arbor, MI, USA. Electronic address:

Craniosynostosis is a debilitating birth defect characterized by the premature fusion of cranial bones resulting from premature loss of stem cells located in suture tissue between growing bones. Mesenchymal stromal cells in long bone and the cranial suture are known to be multipotent cell sources in the appendicular skeleton and cranium, respectively. We are developing biomaterial constructs to maintain stemness of the cranial suture cell population towards an ultimate goal of diminishing craniosynostosis patient morbidity. Recent evidence suggests that physical features of synthetic tissue engineering scaffolds modulate cell and tissue fate. In this study, macroporous tissue engineering scaffolds with well-controlled spherical pores were fabricated by a sugar porogen template method. Cell-scaffold constructs were implanted subcutaneously in mice for up to eight weeks then assayed for mineralization, vascularization, extracellular matrix composition, and gene expression. Pore size differentially regulates cell fate, where sufficiently large pores provide an osteogenic niche adequate for bone formation, while sufficiently small pores (<125 μm in diameter) maintain stemness and prevent differentiation. Cell-scaffold constructs cultured in vitro followed the same pore size-controlled differentiation fate. We therefore attribute the differential cell and tissue fate to scaffold pore geometry. Scaffold pore size regulates mesenchymal cell fate, providing a novel design motif to control tissue regenerative processes and develop mesenchymal stem cell niches in vivo and in vitro through biophysical features.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8068670PMC
May 2021

Human-chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution.

Nat Genet 2021 04 17;53(4):467-476. Epub 2021 Mar 17.

Department of Biology, Stanford University, Stanford, CA, USA.

Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.
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http://dx.doi.org/10.1038/s41588-021-00804-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038968PMC
April 2021

Augmented BMP signaling commits cranial neural crest cells to a chondrogenic fate by suppressing autophagic β-catenin degradation.

Sci Signal 2021 Jan 12;14(665). Epub 2021 Jan 12.

Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI 48109, USA.

Cranial neural crest cells (CNCCs) are a population of multipotent stem cells that give rise to craniofacial bone and cartilage during development. Bone morphogenetic protein (BMP) signaling and autophagy have been individually implicated in stem cell homeostasis. Mutations that cause constitutive activation of the BMP type I receptor ACVR1 cause the congenital disorder fibrodysplasia ossificans progressiva (FOP), which is characterized by ectopic cartilage and bone in connective tissues in the trunk and sometimes includes ectopic craniofacial bones. Here, we showed that enhanced BMP signaling through the constitutively activated ACVR1 (ca-ACVR1) in CNCCs in mice induced ectopic cartilage formation in the craniofacial region through an autophagy-dependent mechanism. Enhanced BMP signaling suppressed autophagy by activating mTORC1, thus blocking the autophagic degradation of β-catenin, which, in turn, caused CNCCs to adopt a chondrogenic identity. Transient blockade of mTORC1, reactivation of autophagy, or suppression of Wnt-β-catenin signaling reduced ectopic cartilages in ca mutants. Our results suggest that BMP signaling and autophagy coordinately regulate β-catenin activity to direct the fate of CNCCs during craniofacial development. These findings may also explain why some patients with FOP develop ectopic bones through endochondral ossification in craniofacial regions.
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http://dx.doi.org/10.1126/scisignal.aaz9368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936468PMC
January 2021

ALK3 Is Not Required for the Embryonic Development, Homeostasis, and Repopulation of Epidermal Langerhans Cells in Steady and Inflammatory States.

J Invest Dermatol 2020 Dec 23. Epub 2020 Dec 23.

Center for Cutaneous Biology and Immunology, Department of Dermatology, Henry Ford Health System, Detroit, Michigan, USA; Immunology Research Program, Henry Ford Cancer Institute, Henry Ford Health System, Detroit, Michigan, USA; Department of Internal Medicine, Henry Ford Health System, Detroit, Michigan, USA. Electronic address:

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http://dx.doi.org/10.1016/j.jid.2020.10.028DOI Listing
December 2020

LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors.

Clin Cancer Res 2021 Apr 22;27(7):2061-2073. Epub 2020 Dec 22.

Novartis Institutes for Biomedical Research, Cambridge, Massachusetts.

Purpose: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254.

Experimental Design: LXH254 was profiled in biochemical, , and assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF.

Results: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and mutants, but had only modest activity in mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, , LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive.

Conclusions: LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2563DOI Listing
April 2021

Mesenchymal VEGFA induces aberrant differentiation in heterotopic ossification.

Bone Res 2019 Dec 10;7(1):36. Epub 2019 Dec 10.

Department of Surgery, University of Michigan, Ann Arbor, MI, 48109, USA.

Heterotopic ossification (HO) is a debilitating condition characterized by the pathologic formation of ectopic bone. HO occurs commonly following orthopedic surgeries, burns, and neurologic injuries. While surgical excision may provide palliation, the procedure is often burdened with significant intra-operative blood loss due to a more robust contribution of blood supply to the pathologic bone than to native bone. Based on these clinical observations, we set out to examine the role of vascular signaling in HO. Vascular endothelial growth factor A (VEGFA) has previously been shown to be a crucial pro-angiogenic and pro-osteogenic cue during normal bone development and homeostasis. Our findings, using a validated mouse model of HO, demonstrate that HO lesions are highly vascular, and that VEGFA is critical to ectopic bone formation, despite lacking a contribution of endothelial cells within the developing anlagen.
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http://dx.doi.org/10.1038/s41413-019-0075-6DOI Listing
December 2019

Acvr1 deletion in osteoblasts impaired mandibular bone mass through compromised osteoblast differentiation and enhanced sRANKL-induced osteoclastogenesis.

J Cell Physiol 2021 Jun 29;236(6):4580-4591. Epub 2020 Nov 29.

Department of Oral Pathology, Hospital of Stomatology, Jilin University, Changchun, China.

Bone morphogenetic protein (BMP) signaling is well known in bone homeostasis. However, the physiological effects of BMP signaling on mandibles are largely unknown, as the mandible has distinct functions and characteristics from other bones. In this study, we investigated the roles of BMP signaling in bone homeostasis of the mandibles by deleting BMP type I receptor Acvr1 in osteoblast lineage cells with Osterix-Cre. We found mandibular bone loss in conditional knockout mice at the ages of postnatal day 21 and 42 in an age-dependent manner. The decreased bone mass was related to compromised osteoblast differentiation together with enhanced osteoclastogenesis, which was secondary to the changes in osteoblasts in vivo. In vitro study revealed that deletion of Acvr1 in the mandibular bone marrow stromal cells (BMSCs) significantly compromised osteoblast differentiation. When wild type bone marrow macrophages were cocultured with BMSCs lacking Acvr1 both directly and indirectly, both proliferation and differentiation of osteoclasts were induced as evidenced by an increase of multinucleated cells, compared with cocultured with control BMSCs. Furthermore, we demonstrated that the increased osteoclastogenesis in vitro was at least partially due to the secretion of soluble receptor activator of nuclear factor-κB ligand (sRANKL), which is probably the reason for the mandibular bone loss in vivo. Overall, our results proposed that ACVR1 played essential roles in maintaining mandibular bone homeostasis through osteoblast differentiation and osteoblast-osteoclast communication via sRANKL.
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http://dx.doi.org/10.1002/jcp.30183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048423PMC
June 2021

Molecular and Cellular Pathogenesis of Ellis-van Creveld Syndrome: Lessons from Targeted and Natural Mutations in Animal Models.

J Dev Biol 2020 Oct 9;8(4). Epub 2020 Oct 9.

Department of Biologic and Materials Sciences & Prosthodontics, School of Dentistry, University of Michigan, Ann Arbor, MI 48109-1078, USA.

Ellis-van Creveld syndrome (EVC; MIM ID #225500) is a rare congenital disease with an occurrence of 1 in 60,000. It is characterized by remarkable skeletal dysplasia, such as short limbs, ribs and polydactyly, and orofacial anomalies. With two of three patients first noted as being offspring of consanguineous marriage, this autosomal recessive disease results from mutations in one of two causative genes: or . The recent identification and manipulation of genetic homologs in animals has deepened our understanding beyond human case studies and provided critical insight into disease pathogenesis. This review highlights the utility of animal-based studies of EVC by summarizing: (1) molecular biology of EVC and EVC2/LIMBIN, (2) human disease signs, (3) dysplastic limb development, (4) craniofacial anomalies, (5) tooth anomalies, (6) tracheal cartilage abnormalities, and (7) EVC-like disorders in non-human species.
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http://dx.doi.org/10.3390/jdb8040025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711556PMC
October 2020

Small molecule inhibition of non-canonical (TAK1-mediated) BMP signaling results in reduced chondrogenic ossification and heterotopic ossification in a rat model of blast-associated combat-related lower limb trauma.

Bone 2020 10 2;139:115517. Epub 2020 Jul 2.

Regenerative Medicine Department, Naval Medical Research Center, Silver Spring, MD, United States of America; Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, United States of America. Electronic address:

Heterotopic ossification (HO) is defined as ectopic bone formation around joints and in soft tissues following trauma, particularly blast-related extremity injuries, thermal injuries, central nerve injuries, or orthopaedic surgeries, leading to increased pain and diminished quality of life. Current treatment options include pharmacotherapy with non-steroidal anti-inflammatory drugs, radiotherapy, and surgical excision, but these treatments have limited efficacy and have associated complication profiles. In contrast, small molecule inhibitors have been shown to have higher specificity and less systemic cytotoxicity. Previous studies have shown that bone morphogenetic protein (BMP) signaling and downstream non-canonical (SMAD-independent) BMP signaling mediated induction of TGF-β activated kinase-1 (TAK1) contributes to HO. In the current study, small molecule inhibition of TAK1, NG-25, was evaluated for its efficacy in limiting ectopic bone formation following a rat blast-associated lower limb trauma and a murine burn tenotomy injury model. A significant decrease in total HO volume in the rat blast injury model was observed by microCT imaging with no systemic complications following NG-25 therapy. Furthermore, tissue-resident mesenchymal progenitor cells (MPCs) harvested from rats treated with NG-25 demonstrated decreased proliferation, limited osteogenic differentiation capacity, and reduced gene expression of Tac1, Col10a1, Ibsp, Smad3, and Sox2 (P < 0.05). Single cell RNA-sequencing of murine cells harvested from the injury site in a burn tenotomy injury model showed increased expression of these genes in MPCs during stages of chondrogenic differentiation. Additional in vitro cell cultures of murine tissue-resident MPCs and osteochondrogenic progenitors (OCPs) treated with NG-25 demonstrated reduced chondrogenic differentiation by 10.2-fold (P < 0.001) and 133.3-fold (P < 0.001), respectively, as well as associated reduction in chondrogenic gene expression. Induction of HO in Tak1 knockout mice demonstrated a 7.1-fold (P < 0.001) and 2.7-fold reduction (P < 0.001) in chondrogenic differentiation of murine MPCs and OCPs, respectively, with reduced chondrogenic gene expression. Together, our in vivo models and in vitro cell culture studies demonstrate the importance of TAK1 signaling in chondrogenic differentiation and HO formation and suggest that small molecule inhibition of TAK1 is a promising therapy to limit the formation and progression of HO.
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http://dx.doi.org/10.1016/j.bone.2020.115517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7945876PMC
October 2020

Controversy of physiological vs. pharmacological effects of BMP signaling: Constitutive activation of BMP type IA receptor-dependent signaling in osteoblast lineage enhances bone formation and resorption, not affecting net bone mass.

Bone 2020 09 27;138:115513. Epub 2020 Jun 27.

Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI 48109-1078, USA. Electronic address:

Bone morphogenetic proteins (BMPs) were first described over 50 years ago as potent inducers of ectopic bone formation when administrated subcutaneously. Preclinical studies have extensively examined the osteoinductive properties of BMPs in vitro and new bone formation in vivo. BMPs (BMP-2, BMP-7) have been used in orthopedics over 15 years. While osteogenic function of BMPs has been widely accepted, our previous studies demonstrated that loss-of-function of BMP receptor type IA (BMPR1A), a potent receptor for BMP-2, increased net bone mass by significantly inhibiting bone resorption in mice, indicating a positive role of BMP signaling in bone resorption. The physiological role of BMPs (i.e. osteogenic vs. osteoclastogenic) is still largely unknown. The purpose of this study was to investigate the physiological role of BMP signaling in endogenous long bones during adult stages. For this purpose, we conditionally and constitutively activated the Smad-dependent canonical BMP signaling thorough BMPR1A in osteoblast lineage cells using the mutant mice (Col1CreER™:caBmpr1a). Because trabecular bones were largely increased in the loss-of-function mouse study for BMPR1A, we hypothesized that the augmented BMP signaling would affect endogenous trabecular bones. In the mutant bones, the Smad phosphorylation was enhanced within physiological level three-fold while the resulting gross morphology, bodyweights, bone mass/shape/length, serum calcium/phosphorus levels, collagen cross-link patterns, and healing capability were all unchanged. Interestingly, we found; 1) increased expressions of both bone formation and resorption markers in femoral bones, 2) increased osteoblast and osteoclast numbers together with dynamic bone formation parameters by trabecular bone histomorphometry, 3) modest bone architectural phenotype with reduced bone quality (i.e. reduced trabecular bone connectivity, larger diametric size but reduced cortical bone thickness, and reduced bone mechanical strength), and 4) increased expression of SOST, a downstream target of the Smad-dependent BMPR1A signaling, in the mutant bones. This study is clinically insightful because gain-of-function of BMP signaling within a physiological window does not increase bone mass while it alters molecular and cellular aspects of osteoblast and osteoclast functions as predicted. These findings help explain the high-doses of BMPs (i.e. pharmacological level) in clinical settings required to substantially induce a bone formation, concurrent with potential unexpected side effects (i.e. bone resorption, inflammation) presumably due to a broader population of cell-types exposed to the high-dose BMPs rather than osteoblastic lineage cells.
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http://dx.doi.org/10.1016/j.bone.2020.115513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423725PMC
September 2020

Energy metabolism: A newly emerging target of BMP signaling in bone homeostasis.

Bone 2020 09 5;138:115467. Epub 2020 Jun 5.

Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Energy metabolism is the process of generating energy (i.e. ATP) from nutrients. This process is indispensable for cell homeostasis maintenance and responses to varying conditions. Cells require energy for growth and maintenance and have evolved to have multiple pathways to produce energy. Both genetic and functional studies have demonstrated that energy metabolism, such as glucose, fatty acid, and amino acid metabolism, plays important roles in the formation and function of bone cells including osteoblasts, osteocytes, and osteoclasts. Dysregulation of energy metabolism in bone cells consequently disturbs the balance between bone formation and bone resorption. Metabolic diseases have also been reported to affect bone homeostasis. Bone morphogenic protein (BMP) signaling plays critical roles in regulating the formation and function of bone cells, thus affecting bone development and homeostasis. Mutations of BMP signaling-related genes in mice have been reported to show abnormalities in energy metabolism in many tissues, including bone. In addition, BMP signaling correlates with critical signaling pathways such as mTOR, HIF, Wnt, and self-degradative process autophagy to coordinate energy metabolism and bone homeostasis. These findings will provide a newly emerging target of BMP signaling and potential therapeutic strategies and the improved management of bone diseases. This review summarizes the recent advances in our understanding of (1) energy metabolism in regulating the formation and function of bone cells, (2) function of BMP signaling in whole body energy metabolism, and (3) mechanistic interaction of BMP signaling with other signaling pathways and biological processes critical for energy metabolism and bone homeostasis.
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http://dx.doi.org/10.1016/j.bone.2020.115467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423769PMC
September 2020

Loss of BMP signaling mediated by BMPR1A in osteoblasts leads to differential bone phenotypes in mice depending on anatomical location of the bones.

Bone 2020 08 1;137:115402. Epub 2020 May 1.

Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, MI, USA. Electronic address:

Bone morphogenetic protein (BMP) signaling in osteoblasts plays critical roles in skeletal development and bone homeostasis. Our previous studies showed loss of function of BMPR1A, one of the type 1 receptors for BMPs, in osteoblasts results in increased trabecular bone mass in long bones due to an imbalance between bone formation and bone resorption. Decreased bone resorption was associated with an increased mature-to-immature collagen cross-link ratio and mineral-matrix ratios in the trabecular compartments, and increased tissue-level biomechanical properties. Here, we investigated the bone mass, bone composition and biomechanical properties of ribs and spines in the same genetically altered mouse line to compare outcomes by loss of BMPR1A functions in bones from different anatomic sites and developmental origins. Bone mass was significantly increased in both cortical and trabecular compartments of ribs with minimal to modest changes in compositions. While tissue-levels of biomechanical properties were not changed between control and mutant animals, whole bone levels of biomechanical properties were significantly increased in association with increased bone mass in the mutant ribs. For spines, mutant bones showed increased bone mass in both cortical and trabecular compartments with an increase of mineral content. These results emphasize the differential role of BMP signaling in osteoblasts in bones depending on their anatomical locations, functional loading requirements and developmental origin.
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http://dx.doi.org/10.1016/j.bone.2020.115402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354232PMC
August 2020

BMP-Smad Signaling Regulates Postnatal Crown Dentinogenesis in Mouse Molar.

JBMR Plus 2020 Feb 14;4(2):e10249. Epub 2019 Nov 14.

Department of Biologic and Materials Sciences and Prosthodontics University of Michigan School of Dentistry Ann Arbor MI USA.

Dentinogenesis, a formation of dentin by odontoblasts, is an essential process during tooth development. Bone morphogenetic proteins (BMPs) are one of the most crucial growth factors that contribute to dentin formation. However, it is still unclear how BMP signaling pathways regulate postnatal crown and root dentinogenesis. BMPs transduce signals through canonical Smad and non-Smad signaling pathways including p38 and ERK signaling pathways. To investigate the roles of Smad and non-Smad signaling pathways in dentinogenesis, we conditionally deleted , which encodes the type 1A receptor for BMPs, to remove both Smad and non-Smad pathways in -expressing cells. We also expressed a constitutively activated form of () to increase Smad1/5/9 signaling activity without altered non-Smad activity in odontoblasts. To understand the function of BMP signaling during postnatal dentin formation, Cre activity was induced at the day of birth. Our results showed that loss of BmpR1A in odontoblasts resulted in impaired dentin formation and short molar roots at postnatal day 21. cKO mice displayed a reduction of dentin matrix production compared to controls associated with increased cell proliferation and reduced and expression. In contrast, mutant mice that show increased Smad1/5/9 signaling activity resulted in no overt tooth phenotype. To further dissect the functions of each signaling activity, we generated cKO mice also expressing to restore only Smad1/5/9 signaling activity. Restoring Smad activity in the compound mutant mice rescued impaired crown dentin formation in the cKO mice; however, impaired root dentin formation and short roots were not changed. These results suggest that BMP-Smad signaling in odontoblasts is responsible for crown dentin formation, while non-Smad signaling may play a major role in root dentin formation and elongation. © 2019 The Authors. published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research. © 2019 The Authors. published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017888PMC
February 2020

Methods for the reliable induction of heterotopic ossification in the conditional mouse.

J Musculoskelet Neuronal Interact 2020 03;20(1):149-159

Vanderbilt Center for Bone Biology.

Objectives: Conditional () mice have previously been used as a model of heterotopic ossification (HO). However, HO formation in this model can be highly variable, and it is unclear which methods reliably induce HO. Hence, these studies report validated methods for reproducibly inducing HO in mice.

Methods: Varying doses of Adex-cre and cardiotoxin (CTX) were injected into the calf muscles of 9, 14, or 28-day-old or mice. HO was measured by planar radiography or microCT at 14-28 days post-injury.

Results: In 9-day-old or mice, single injections of 10 PFU Adex-cre and 0.3 μg of CTX were sufficient to induce extensive HO within 14 days post-injury. In 28-day-old mice, the doses were increased to 5 x 10 PFU Adex-cre and 3.0 μg of CTX to achieve similar consistency, but at a slower rate versus younger mice. Using a crush injury, instead of CTX, also provided consistent induction of HO. Finally, the Type 1 BMPR inhibitor, DMH1, significantly reduced HO formation in 28-day-old mice.

Conclusions: These data illustrate multiple methods for reliable induction of localized HO in the mouse that can serve as a starting point for new laboratories utilizing this model.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104591PMC
March 2020

Regulation of heterotopic ossification by monocytes in a mouse model of aberrant wound healing.

Nat Commun 2020 02 5;11(1):722. Epub 2020 Feb 5.

Section of Plastic Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI, 48109, USA.

Heterotopic ossification (HO) is an aberrant regenerative process with ectopic bone induction in response to musculoskeletal trauma, in which mesenchymal stem cells (MSC) differentiate into osteochondrogenic cells instead of myocytes or tenocytes. Despite frequent cases of hospitalized musculoskeletal trauma, the inflammatory responses and cell population dynamics that regulate subsequent wound healing and tissue regeneration are still unclear. Here we examine, using a mouse model of trauma-induced HO, the local microenvironment of the initial post-injury inflammatory response. Single cell transcriptome analyses identify distinct monocyte/macrophage populations at the injury site, with their dynamic changes over time elucidated using trajectory analyses. Mechanistically, transforming growth factor beta-1 (TGFβ1)-producing monocytes/macrophages are associated with HO and aberrant chondrogenic progenitor cell differentiation, while CD47-activating peptides that reduce systemic macrophage TGFβ levels and help ameliorate HO. Our data thus implicate CD47 activation as a therapeutic approach for modulating monocyte/macrophage phenotypes, MSC differentiation and HO formation during wound healing.
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http://dx.doi.org/10.1038/s41467-019-14172-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002453PMC
February 2020

Mesenchymal VEGFA induces aberrant differentiation in heterotopic ossification.

Bone Res 2019 10;7:36. Epub 2019 Dec 10.

1Department of Surgery, University of Michigan, Ann Arbor, MI 48109 USA.

Heterotopic ossification (HO) is a debilitating condition characterized by the pathologic formation of ectopic bone. HO occurs commonly following orthopedic surgeries, burns, and neurologic injuries. While surgical excision may provide palliation, the procedure is often burdened with significant intra-operative blood loss due to a more robust contribution of blood supply to the pathologic bone than to native bone. Based on these clinical observations, we set out to examine the role of vascular signaling in HO. Vascular endothelial growth factor A (VEGFA) has previously been shown to be a crucial pro-angiogenic and pro-osteogenic cue during normal bone development and homeostasis. Our findings, using a validated mouse model of HO, demonstrate that HO lesions are highly vascular, and that VEGFA is critical to ectopic bone formation, despite lacking a contribution of endothelial cells within the developing anlagen.
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http://dx.doi.org/10.1038/s41413-019-0075-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904752PMC
December 2019

The noncanonical BMP signaling pathway plays an important role in club cell regeneration.

Stem Cells 2020 03 6;38(3):437-450. Epub 2019 Dec 6.

Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, People's Republic of China.

The bronchiole is a major site for the development of several life-threatening disorders, including chronic obstructive pulmonary disease and lung adenocarcinomas. The bronchiolar epithelium is composed of club cells and ciliated epithelial cells, with club cells serving as progenitor cells. Presently, the identity of the cells involved in regeneration of bronchiolar epithelium and the underlying mechanisms remain incompletely understood. Here, we show that Prrx1, a homeobox transcription factor, can mark club cells in adult mice during homeostasis and regeneration. We further show that the noncanonical signaling pathway of BMPs, BMPR1A-Tak1-p38MAPK, plays a critical role in club cell regeneration. Ablation of Bmpr1a, Tak1, or Mapk14 (encoding p38α) in Prrx1 club cells caused minimal effect on bronchiolar epithelium homeostasis, yet it resulted in severe defects in club cell regeneration and bronchiole repair in adult mice. We further show that this pathway supports proliferation and expansion of the regenerating club cells. Our findings thus identify a marker for club cells and reveal a critical role for the BMP noncanonical pathway in club cell regeneration.
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http://dx.doi.org/10.1002/stem.3125DOI Listing
March 2020

Disruption of Neutrophil Extracellular Traps (NETs) Links Mechanical Strain to Post-traumatic Inflammation.

Front Immunol 2019 24;10:2148. Epub 2019 Oct 24.

Department of Surgery, University of Michigan Medical School, Ann Arbor, MI, United States.

Inflammation after trauma is both critical to normal wound healing and may be highly detrimental when prolonged or unchecked with the potential to impair physiologic healing and promote pathology. Mechanical strain after trauma is associated with impaired wound healing and increased inflammation. The exact mechanisms behind this are not fully elucidated. Neutrophil extracellular traps (NETs), a component of the neutrophil response to trauma, are implicated in a range of pro-inflammatory conditions. In the current study, we evaluated their role in linking movement and inflammation. We found that a link exists between the disruption and amplification of NETs which harbors the potential to regulate the wound's response to mechanical strain, while leaving the initial inflammatory signal necessary for physiologic wound healing intact.
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http://dx.doi.org/10.3389/fimmu.2019.02148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821718PMC
October 2020

Activin A receptor type 1-mediated BMP signaling regulates RANKL-induced osteoclastogenesis via canonical SMAD-signaling pathway.

J Biol Chem 2019 11 16;294(47):17818-17836. Epub 2019 Oct 16.

Department of Biologic and Materials Sciences and Prosthodontics, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109

Bone morphogenetic proteins (BMPs) are important mediators of osteoclast differentiation. Although accumulating evidence has implicated BMPs in osteoblastogenesis, the mechanisms by which BMPs regulate osteoclastogenesis remain unclear. Activin A receptor type 1 (ACVR1) is a BMP type 1 receptor essential for skeletal development. Here, we observed that BMP-7, which preferentially binds to ACVR1, promotes osteoclast differentiation, suggesting ACVR1 is involved in osteoclastogenesis. To investigate this further, we isolated osteoclasts from either -floxed mice or mice with constitutively-activated () carrying tamoxifen-inducible Cre driven by a ubiquitin promotor and induced Cre activity in culture. Osteoclasts from the -floxed mice had reduced osteoclast numbers and demineralization activity, whereas those from the -mutant mice formed large osteoclasts and demineralized pits, suggesting that BMP signaling through ACVR1 regulates osteoclast fusion and activity. It is reported that BMP-2 binds to BMPR1A, another BMP type 1 receptor, whereas BMP-7 binds to ACVR1 to activate SMAD1/5/9 signaling. Here, -disrupted osteoclasts displayed reduced phospho-SMAD1/5/9 (pSMAD1/5/9) levels when induced by BMP-2, whereas no impacts on pSMAD1/5/9 were observed when induced by BMP-7. In contract, -disrupted osteoclasts displayed reduced pSMAD1/5/9 levels when induced either by BMP-2 or BMP-7, suggesting that ACVR1 is the major receptor for transducing BMP-7 signals in osteoclasts. Indeed, LDN-193189 and LDN-212854, which specifically block SMAD1/5/9 phosphorylation, inhibited osteoclastogenesis of -mutant cells. Moreover, increased BMP signaling promoted nuclear translocation of nuclear factor-activated T-cells 1 (NFATc1), which was inhibited by LDN treatments. Taken together, ACVR1-mediated BMP-SMAD signaling activates NFATc1, a regulatory protein crucial for receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis.
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http://dx.doi.org/10.1074/jbc.RA119.009521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879329PMC
November 2019

Covalent Attachment of P15 Peptide to Ti Alloy Surface Modified with Polymer to Enhance Osseointegration of Implants.

ACS Appl Mater Interfaces 2019 Oct 10;11(42):38531-38536. Epub 2019 Oct 10.

Department of Chemistry , Carnegie Mellon University , Pittsburgh , Pennsylvania 15213 , United States.

Titanium (Ti) and its alloys are used in orthopedic and dental implants due to their excellent physical properties and biocompatibility. Although Ti exhibits superior osteoconductive properties compared to those of polymer-based implants, improved bone-on growth properties are required for enhanced surgical outcomes and improved recovery surgical interventions. Herein, we demonstrate a novel surface modification strategy to enhance the osteoconductivity of Ti surfaces through the grafting-from procedure of a reactive copolymer via surface-initiated atom transfer radical polymerization (SI-ATRP). Then, postpolymerization conjugation of the P15 peptide, an osteoblast binding motif, was successfully carried out. Subsequent studies revealed that the surface modification promoted osteoblast attachment on the Ti discs at 6 and 24 h. Moreover, mineral matrix deposition by osteoblasts was greater for the surface-modified Ti than for plain Ti and P15 randomly absorbed onto the Ti surface. These results suggest that the strategy for postpolymerization incorporation of P15 onto a Ti surface with a polymer interface may provide improved osseointegration outcomes, leading to enhanced quality of life for patients.
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http://dx.doi.org/10.1021/acsami.9b14651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993989PMC
October 2019

Increased activity of mesenchymal ALK2-BMP signaling causes posteriorly truncated microglossia and disorganization of lingual tissues.

Genesis 2020 01 30;58(1):e23337. Epub 2019 Sep 30.

Department of Animal and Dairy Science, Regenerative Bioscience Center, College of Agricultural and Environmental Sciences, University of Georgia, Athens, Georgia.

Proper development of taste organs including the tongue and taste papillae requires interactions with the underlying mesenchyme through multiple molecular signaling pathways. The effects of bone morphogenetic proteins (BMPs) and antagonists are profound, however, the tissue-specific roles of distinct receptors are largely unknown. Here, we report that constitutive activation (ca) of ALK2-BMP signaling in the tongue mesenchyme (marked by Wnt1-Cre) caused microglossia-a dramatically smaller and misshapen tongue with a progressively severe reduction in size along the anteroposterior axis and absence of a pharyngeal region. At E10.5, the tongue primordia (branchial arches 1-4) formed in Wnt1-Cre/caAlk2 mutants while each branchial arch responded to elevated BMP signaling distinctly in gene expression of BMP targets (Id1, Snai1, Snai2, and Runx2), proliferation (Cyclin-D1) and apoptosis (p53). Moreover, elevated ALK2-BMP signaling in the mesenchyme resulted in apparent defects of lingual epithelium, muscles, and nerves. In Wnt1-Cre/caAlk2 mutants, a circumvallate papilla was missing and further development of formed fungiform papillae was arrested in late embryos. Our data collectively demonstrate that ALK2-BMP signaling in the mesenchyme plays essential roles in orchestrating various tissues for proper development of the tongue and its appendages in a region-specific manner.
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http://dx.doi.org/10.1002/dvg.23337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980365PMC
January 2020

Inhibition of phosphatidylinositol 3-kinase α (PI3Kα) prevents heterotopic ossification.

EMBO Mol Med 2019 09 2;11(9):e10567. Epub 2019 Aug 2.

Departament de Ciències Fisiològiques, Universitat de Barcelona, IDIBELL, Hospitalet de Llobregat, Spain.

Heterotopic ossification (HO) is the pathological formation of ectopic endochondral bone within soft tissues. HO occurs following mechanical trauma, burns, or congenitally in patients suffering from fibrodysplasia ossificans progressiva (FOP). FOP patients carry a conserved mutation in ACVR1 that becomes neomorphic for activin A responses. Here, we demonstrate the efficacy of BYL719, a PI3Kα inhibitor, in preventing HO in mice. We found that PI3Kα inhibitors reduce SMAD, AKT, and mTOR/S6K activities. Inhibition of PI3Kα also impairs skeletogenic responsiveness to BMPs and the acquired response to activin A of the Acvr1 allele. Further, the efficacy of PI3Kα inhibitors was evaluated in transgenic mice expressing Acvr1 . Mice treated daily or intermittently with BYL719 did not show ectopic bone or cartilage formation. Furthermore, the intermittent treatment with BYL719 was not associated with any substantial side effects. Therefore, this work provides evidence supporting PI3Kα inhibition as a therapeutic strategy for HO.
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http://dx.doi.org/10.15252/emmm.201910567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728602PMC
September 2019

Autophagy Regulates Craniofacial Bone Acquisition.

Calcif Tissue Int 2019 11 1;105(5):518-530. Epub 2019 Aug 1.

Department of Biologic and Materials Sciences & Prosthodontics, University of Michigan School of Dentistry, Ann Arbor, MI, 48109, USA.

Increasing evidence has demonstrated the important role of autophagy in skeletal homeostasis; however, the role of autophagy in craniofacial bone development and acquisition is largely unknown. In this study, we investigated the effect of autophagy suppression on craniofacial bone acquisition by deleting Fip200 or Atg5, two essential autophagy genes, using Osterix-Cre (Osx-Cre). We found that the Osx-Cre transgene mildly decreased the bone mass of parietal bone but not frontal bone, and did not affect cranial base bone mass in adult mice. In the cranial vault, Fip200 or Atg5 deletion similarly decreased 50% bone mass of neural crest-derived frontal bone; Atg5 deletion decreased 50% and Fip200 deletion decreased 30% bone mass of mesoderm-derived parietal bone. In the cranial base, Fip200 or Atg5 deletion similarly decreased 30% bone mass of neural crest-derived presphenoid bone; Atg5 deletion decreased 30% and Fip200 deletion decreased 16% bone mass of mesoderm-derive basioccipital bone. Lastly, we used doxycycline treatment to inhibit the Osx-Cre expression until 2 months of age and showed that postnatal Fip200 deletion led to cranial vault bone mass decrease in association with a small increase in both bone volume/tissue volume and tissue mineral density. Altogether, this study demonstrated the important role of autophagy in craniofacial bone acquisition during development and postnatal growth.
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http://dx.doi.org/10.1007/s00223-019-00593-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801085PMC
November 2019

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone.

J Vis Exp 2019 05 10(147). Epub 2019 May 10.

Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan;

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.
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http://dx.doi.org/10.3791/59113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996940PMC
May 2019

Design and Discovery of -(3-(2-(2-Hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide, a Selective, Efficacious, and Well-Tolerated RAF Inhibitor Targeting RAS Mutant Cancers: The Path to the Clinic.

J Med Chem 2020 03 16;63(5):2013-2027. Epub 2019 May 16.

Oncology, Novartis Institutes for BioMedical Research, 5300 Chiron Way, Emeryville, California 94608, United States.

Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective B/C RAF inhibitor, which was developed by focusing on drug-like properties and selectivity. Our previous tool compound, (RAF709; Nishiguchi, G. A.; et al. , , 4969), was potent, selective, efficacious, and well tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further investigation of close analogues. A structure-based approach led to a pyridine series with an alcohol side chain that could interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition led to the discovery of . Due to its excellent properties, it was progressed through toxicology studies and is being tested in phase 1 clinical trials.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00161DOI Listing
March 2020

Trim33 is required for appropriate development of pre-cardiogenic mesoderm.

Dev Biol 2019 06 30;450(2):101-114. Epub 2019 Mar 30.

Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, MI, 48109, USA. Electronic address:

Congenital cardiac malformations are among the most common birth defects in humans. Here we show that Trim33, a member of the Tif1 subfamily of tripartite domain containing transcriptional cofactors, is required for appropriate differentiation of the pre-cardiogenic mesoderm during a narrow time window in late gastrulation. While mesoderm-specific Trim33 mutants did not display noticeable phenotypes, epiblast-specific Trim33 mutant embryos developed ventricular septal defects, showed sparse trabeculation and abnormally thin compact myocardium, and died as a result of cardiac failure during late gestation. Differentiating embryoid bodies deficient in Trim33 showed an enrichment of gene sets associated with cardiac differentiation and contractility, while the total number of cardiac precursor cells was reduced. Concordantly, cardiac progenitor cell proliferation was reduced in Trim33-deficient embryos. ChIP-Seq performed using antibodies against Trim33 in differentiating embryoid bodies revealed more than 4000 peaks, which were significantly enriched close to genes implicated in stem cell maintenance and mesoderm development. Nearly half of the Trim33 peaks overlapped with binding sites of the Ctcf insulator protein. Our results suggest that Trim33 is required for appropriate differentiation of precardiogenic mesoderm during late gastrulation and that it will likely mediate some of its functions via multi-protein complexes, many of which include the chromatin architectural and insulator protein Ctcf.
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http://dx.doi.org/10.1016/j.ydbio.2019.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547372PMC
June 2019

Coordinating Tissue Regeneration Through Transforming Growth Factor-β Activated Kinase 1 Inactivation and Reactivation.

Stem Cells 2019 06 14;37(6):766-778. Epub 2019 Mar 14.

School of Dentistry, University of Michigan, Ann Arbor, Michigan, USA.

Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of transforming growth factor-β activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by reactivation to elicit differentiation and extracellular matrix production. Although the current regenerative medicine paradigm is centered on the effects of drug treatment ("drug on"), the impact of drug withdrawal ("drug off") implicit in these regimens is unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment ("drug on") and subsequent withdrawal ("drug off") through genetic modification, was used here to inactivate and reactivate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the "drug on" (Cre-mediated inactivation) and "drug off" (Flp-mediated reactivation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. Stem Cells 2019;37:766-778.
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http://dx.doi.org/10.1002/stem.2991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542699PMC
June 2019