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36


Membrane-type 1 Matrix Metalloproteinase Modulates Tissue Homeostasis by a Non-proteolytic Mechanism

Attur, Mukundan; Lu, Cuijie; Zhang, Xiaodong; Han, Tianzhen; Alexandre, Cassidy; Valacca, Cristina; Zheng, Shuai; Meikle, Sarina; Dabovic, Branka Brukner; Tassone, Evelyne; Yang, Qing; Kolupaeva, Victoria; Yakar, Shoshana; Abramson, Steven; Mignatti, Paolo
Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with a short cytoplasmic tail, is a major effector of extracellular matrix remodeling. Genetic silencing of MT1-MMP in mouse (Mmp14
PMCID:7695985
PMID: 33294797
ISSN: 2589-0042
CID: 4735172

Abaloparatide at the same dose has the same effects on bone as PTH (1-34) in mice

Le Henaff, Carole; Ricarte, Florante; Finnie, Brandon; He, Zhiming; Johnson, Joshua; Warshaw, Johanna; Kolupaeva, Victoria; Partridge, Nicola C
Abaloparatide, a novel analog of parathyroid hormone-related protein (PTHrP 1-34) became, in 2017, the second osteoanabolic therapy for the treatment of osteoporosis. This study aims to compare the effects of PTH (1-34), PTHrP (1-36), and abaloparatide on bone remodeling in male mice. Intermittent daily subcutaneous injections of 80 μg/kg/day were administered to four-month-old C57Bl/6J male mice for six weeks. During treatment, mice were followed by DEXA-Piximus to assess changes in bone mineral density (BMD) in the whole body, femur and tibia. At either four or eighteen hours after the final injection, femurs were harvested for μCT analyses and histomorphometry, sera were assayed for bone turnover marker levels, and tibiae were separated into cortical, trabecular, and bone marrow fractions for gene expression analyses. Our results showed that, compared with PTH (1-34), abaloparatide resulted in a similar increase in BMD at all sites, while no changes were seen with PTHrP (1-36). With both PTH (1-34) and abaloparatide, μCT and histomorphometry analyses revealed similar increases in bone volume associated with an increased trabecular thickness, in bone formation rate as shown by P1NP serum level and in vivo double labeling, and in bone resorption as shown by CTX levels and osteoclast number. Gene expression analyses of trabecular and cortical bone showed that PTH (1-34) and abaloparatide led to different actions in osteoblast differentiation and activity, with increased Runx2, Col1A1, Alpl, Bsp, Ocn, Sost, Rankl/Opg and c-fos at different time points. Abaloparatide seems to generate a faster response on osteoblastic gene expression than PTH (1-34). Taken together, abaloparatide at the same dose is as effective as PTH (1-34) as an osteoanabolic, with an increase in bone formation but also an increase in bone resorption in male mice. This article is protected by copyright. All rights reserved.
PMID: 31793033
ISSN: 1523-4681
CID: 4218282

Translational control maintains cartilage homeostasis and regulates osteoarthritis progression [Meeting Abstract]

Kolupaeva, V; Katsara, O; Attur, M
Purpose: The unique properties of articular cartilage are defined by quiescent, differentiated articular chondrocytes whose homeostasis is maintained through a strict balance between anabolic and catabolic processes. This balance is shifted toward catabolic activities in osteoarthritis (OA), a disease that involves all joint tissues and disrupts the structural integrity of articular cartilage. In OA, articular chondrocytes become more hypertrophic-ike, and abnormally increase not only extracellular matrix (ECM) production but also the expression of ECM-degrading enzymes. While chondrocyte hypertrophy is indispensable for endochondral bone formation, in OA it mediates detrimental changes that lead to cartilage destruction. Our previous work established that translational control of gene expression is critical for dedifferentiation of articular chondrocytes in OA. We showed that fine-tuned control of protein synthesis by translational repressor 4E-BP (eIF4E-Binding Protein) is required for proper chondrocyte homeostasis. By binding to the m7-GTP cap-binding protein eIF4E, 4E-BP inhibits cap-dependent mRNA translation by restricting the incorporation of eIF4E into a multi-subunit initiation factor called eIF4F, which is needed to recruit 40S ribosomes to mRNA. We hypothesized that proper translational control is crucial for maintaining healthy cartilage phenotype. We therefore investigated expression of which genes is controlled (completely or partially) at the translational level. Method(s): To identify the pool of mRNAs that are translationally control in OA cartilage we first compared translation efficiencies of mRNAs by analyzing polysome profiles of rat articular chondrocytes (RAC) untreated and treated with Interleukin 1beta (IL-1beta). Expression of several translationally regulated mRNAs was then compared to their expression in human and rodent OA cartilage. Additionally, the eIF4F inhibitor was used in vivo to determine if restriction of eIF4F activity affects OA progression in ACLT rat model. Result(s): We identified 617 mRNAs that are translationally regulated by IL-1beta. One of them is the orphan receptor Nr4a1, whose protein but not mRNA level is significantly increased in OA cartilage in vivo, highlighting the exceptional potential of our approach for discovering new possible targets in OA cartilage. Modulating Nr4a1 expression in IL-1beta-treated articular chondrocytes effects expression of MMP13, the metalloproteinase that has a predominant role in OA. Intra-articular injection of 4E1RCat, an inhibitor of cap-dependent translation not only precludes Fn and Nr4a upregulation in a rodent OA model, but also significantly delays cartilage degeneration in the ACLT rat model. Conclusion(s): We identified 617 mRNAs that are translationally regulated by IL-1beta. One of them is the orphan receptor Nr4a1, whose protein but not mRNA level is significantly increased in OA cartilage in vivo, highlighting the exceptional potential of our approach for discovering new possible targets in OA cartilage. Modulating Nr4a1 expression in IL-1beta-treated articular chondrocytes effects expression of MMP13, the metalloproteinase that has a predominant role in OA. Intra-articular injection of 4E1RCat, an inhibitor of cap-dependent translation not only precludes Fn and Nr4a upregulation in a rodent OA model, but also significantly delays cartilage degeneration in the ACLT rat model.
EMBASE:2001663068
ISSN: 1522-9653
CID: 3789892

Serine-threonine protein phosphatases: Lost in translation

Kolupaeva, Victoria
Protein synthesis is one of the most complex and energy-consuming processes in eukaryotic cells and therefore is tightly regulated. One of the main mechanisms of translational control is post-translational modifications of the components of translational apparatus. Phosphorylation status of translation factors depends on the balanced action of kinases and phosphatases. While many kinase-dependent events are well defined, phosphatases that counteract phosphorylation are rarely determined. This mini-review focuses on the regulation of activity of translational initiation factors by serine/threonine phosphatases.
PMID: 30401537
ISSN: 1879-2596
CID: 3491192

Protein Phosphatases 1 and 2A are Necessary for PTH (1-34) Stimulation of Osteoblastic RANKL Expression Through CREB Regulated Transcription Coactivator 3 [Meeting Abstract]

He, Zhiming; Le Henaff, Carole; Ricarte, Florante; Kolupaeva, Victoria; Partridge, Nicola
ISI:000508614701386
ISSN: 0884-0431
CID: 4337652

Parathyroid hormone (1-34) and its analogs differentially modulate osteoblastic RANKL expression via PKA/PP1/PP2A and SIK2/SIK3-CRTC3 signaling

Ricarte, Florante R; Le Henaff, Carole; Kolupaeva, Victoria G; Gardella, Thomas J; Partridge, Nicola C
Osteoporosis can result from the loss of sex hormones and/or aging. Abaloparatide (ABL), an analog of parathyroid hormone-related protein (PTHrP 1-36), is the second osteoanabolic therapy approved by the US Food and Drug Administration after teriparatide (PTH 1-34). All three peptides bind PTH/PTHrP receptor type 1 (PTHR1), but the effects of PTHrP (1-36) or ABL in the osteoblast remain unclear. We show that, in primary calvarial osteoblasts, PTH (1-34) promotes a more robust cAMP response than PTHrP (1-36) and ABL and causes a greater activation of protein kinase A (PKA) and cAMP response element-binding protein (CREB). All three peptides similarly inhibited sclerostin (SOST). Interestingly, the three peptides differentially modulated two other PKA target genes, c-Fos and receptor activator of nuclear factor kappa-B ligand (RANKL), and the latter both in vitro and in vivo. Knockdown of salt-inducible kinases (SIKs) 2 and 3 and CREB-regulated transcription coactivator 3 (CRTC3), indicated that all three are part of the pathway that regulates osteoblastic RANKL expression. We also show that the peptides differentially regulate the nuclear localization of CRTC2 and CRTC3, and that this correlates with PKA activation. Moreover, inhibition of protein phosphatases 1 and 2A (PP1/PP2A) activity revealed that they play a major role in both PTH-induced RANKL expression and the effects of PTH (1-34) on CRTC3 localization. In summary, in the osteoblast, the effects of PTH (1-34), PTHrP (1-36), and ABL on RANKL are mediated by differential stimulation of cAMP/PKA signaling and by their downstream effects on SIK2 and 3, PP1/PP2A, and CRTC3.
PMID: 30377251
ISSN: 1083-351x
CID: 3399712

mTOR-mediated inactivation of 4E-BP1, an inhibitor of translation, precedes cartilage degeneration in rat osteoarthritic knees

Katsara, Olga; Kolupaeva, Victoria
Proper control of protein synthesis is vital for tissue homeostasis and its deregulation is characteristic of many disorders including osteoarthritis (OA). The objectives of this work were to analyze and correlate changes in activity of the translation apparatus associated with cartilage degeneration in an animal model of OA. Osteoarthritis was induced surgically in rats by anterior cruciate ligament transection (ACLT). Using a modified Mankin scoring system and analysis of protein expression we demonstrated, that mechanistic target of rapamycin complex 1 (mTORC1)-mediated 4E-BP1 phosphorylation was detected significantly earlier than other mTORC1-mediated modifications, such as p70S6K and ULK1 phosphorylation. 4E-BP1 is an inhibitor of cap-dependent translation those functions are inhibited by mTORC1 mediated phosphorylation. This signaling event not only preceded prominent signs of cartilage degeneration but also the increase in global protein synthesis rate. These results suggest that abnormal mTORC1 activity is one of the primary dysregulations observed in OA cartilage. Importantly, it is distributed disproportionately between targets, with 4E-BP1 being phosphorylated earlier than other downstream targets. Thus, our work provides new insights into the sequence of molecular events leading to cartilage destruction in OA and identifies translational control as an important regulatory hub involved in initiating OA.
PMID: 29761560
ISSN: 1554-527x
CID: 3121372

THE TRANSLATIONAL LANDSCAPE IN ARTICULAR CHONDROCYTES TREATEDWITH INTERLEUKIN-1 REVEALS NOVEL POTENTIAL PLAYERS IN OSTEOARTHRITIS [Meeting Abstract]

Kolupaeva, V.; Katsara, O.; Attur, M.
ISI:000432189700165
ISSN: 1063-4584
CID: 3132612

Phosphoproteomics of FGF1 signaling in chondrocytes: Identifying the signature of inhibitory response

Chapman, Jessica R; Katsara, Olga; Ruoff, Rachel; Morgenstern, David; Nayak, Shruti; Basilico, Claudio; Ueberheide, Beatrix; Kolupaeva, Victoria
Fibroblast growth factor (FGF) signaling is vital for many biological processes, beginning with development. The importance of FGF signaling for skeleton formation was first discovered by the analysis of genetic FGFR mutations which cause several bone morphogenetic disorders, including achondroplasia, the most common form of human dwarfism. The formation of the long bones is mediated through proliferation and differentiation of highly specialized cells - chondrocytes. Chondrocytes respond to FGF with growth inhibition, a unique response which differs from the proliferative response of the majority of cell types; however its molecular determinants are still unclear. Quantitative phosphoproteomic analysis was utilized to catalogue the proteins whose phosphorylation status is changed upon FGF1 treatment. The generated dataset consists of 756 proteins. We were able to localize the divergence between proliferative (canonical) and inhibitory (chondrocyte specific) FGF transduction pathways immediately upstream of AKT kinase. Gene Ontology (GO) analysis of the FGF1 regulated peptides revealed that many of the identified phosphorylated proteins are assigned to negative regulation clusters, in accordance with the observed inhibitory growth response. This is the first time a comprehensive subset of proteins involved in FGF inhibitory response is defined. We were able to identify a number of targets and specifically discover glycogen synthase kinase3beta (GSK3beta) as a novel key mediator of FGF inhibitory response in chondrocytes.
PMCID:5461542
PMID: 28298517
ISSN: 1535-9484
CID: 2490042

TARGETING A SELECTIVE POOL OF MRNAS TO INTERFERE WITH PROGRESSION OF OSTEOARTHRITIS (OA) [Meeting Abstract]

Katsara, O; Attur, M; Kolupaeva, V
ISI:000406888100257
ISSN: 1522-9653
CID: 2675522