Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes

Abstract

Notch signaling has been identified as a critical regulator of cartilage development and homeostasis. Its pivotal role was established by both several joint specific Notch signaling loss of function mouse models and transient or sustained overexpression. NOTCH1 is the most abundantly expressed NOTCH receptors in normal cartilage and its expression increases in osteoarthritis (OA), when chondrocytes exit from their healthy “maturation arrested state” and resume their natural route of proliferation, hypertrophy, and terminal differentiation. The latter are hallmarks of OA that are easily evaluated in vitro in 2-D or 3-D culture models. The aim of our study was to investigate the effect of NOTCH1 knockdown on proliferation (cell count and Picogreen mediated DNA quantification), cell cycle (flow cytometry), hypertrophy (gene and protein expression of key markers such as RUNX2 and MMP-13), and terminal differentiation (viability measured in 3-D cultures by luminescence assay) of human OA chondrocytes. NOTCH1 silencing of OA chondrocytes yielded a healthier phenotype in both 2-D (reduced proliferation) and 3-D with evidence of decreased hypertrophy (reduced expression of RUNX2 and MMP-13) and terminal differentiation (increased viability). This demonstrates that NOTCH1 is a convenient therapeutic target to attenuate OA progression.

NOTCH1: A Novel Player in the Molecular Crosstalk Underlying Articular Chondrocyte Protection by Oleuropein and Hydroxytyrosol

“…The findings of DAPT effects on JNK > P-c-Jun axis are in keeping with other literature reports that show that “Notch interferes with the scaffold function of JNK-interacting protein 1 to inhibit the JNK signaling pathway” [ 38 ]. Noteworthily, our present data together with previously published results [ 27 , 28 ] would suggest that, in chondrocytes, MMP-13 transcription depends on the combined activity of activated NOTCH1 (cleaved at Val1744, by γ secretase) and phosphorylated c-Jun. In addition, a crosstalk between Notch and NF-κB (noncanonical) signaling pathways has been described as enhancing the transcription of many OA relevant genes, including IL6 and MMP13 [ 35 ].…”

“…We investigated the induction of genes contributing to the amplification of the inflammatory/hypertrophic deregulation of chondrocytes: Inducible Nitric Oxide Synthase ( INOS ), involved in the generation of nitric oxide species and exerting a key role in OA pathogenesis [ 30 ]; Cyclooxygenase-2 ( COX2 ), involved in the production of prostaglandins, mediators of pain [ 31 ]; and NOTCH1 , which we recently showed as pivotal in supporting many pathways of loss of maturational arrest in chondrocytes [ 27 ].…”

“…In conclusion, our study confirms a key role for NOTCH1 in the pathogenesis of OA, thus pointing at this signaling pathway as a potential target for therapy. However, as extensively reviewed elsewhere [ 27 ], NOTCH1 is a kind of double-edged sword with both homeostatic and unwanted effects. Therefore, to avoid the latter, attempts should be made to develop a strategy for preventing excessive activation while sparing its ability to inhibit JNK activation.…”

“…For this study, two in vitro models were used: primary chondrocytes and C28/I2, an immortalized human chondrocyte line [ 17 ]. Human primary chondrocytes ( n = 22) were isolated from knee cartilage derived from OA patients undergoing arthroplasty as previously indicated [ 27 ]. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Istituto Ortopedico Rizzoli (ethic approval code: 0019715, approved on 28 September 2016), including documentation of written patient informed consent.…”

“…Mengshol et al previously reported the requirement of an interaction between Runt-related transcription factor 2 ( RUNX2 ) and AP-1 in MMP-13 transcription [ 26 ]. Recent work from our and other groups has underlined that, in MMP-13 transcription, a fundamental role is also played by Notch homolog 1 (NOTCH1) signaling that promotes RUNX2 activation [ 27 , 28 ]. Since a crosstalk between JNK and the NOTCH1 signaling pathway has been described in macrophages in the regulation of inflammatory responses, with the former activating the latter [ 29 ], we explored whether this crosstalk also occurs in articular chondrocytes.…”