New research points a finger at a previously unknown long noncoding RNA (lncRNA) as playing a critical role in activating nuclear factor kappa B (NF-kappaB) inflammatory responses and driving the progression of osteoarthritis of the knee.

In an in vitro model, downregulating expression of this lncRNA, named PILA (PRTM1-interacting lncRNA), suppressed activation of the NF-kappaB pathway and attenuated cytokine-induced degradation of the extracellular matrix (ECM) in knee cartilage. Conversely, overexpression of PILA promoted activation of the NF-kappaB pathway and accelerated degradation of ECM.

"We believe that our study provides previously unidentified insights into the complex regulation of NF-kappaB signaling and a potentially new therapeutic target," the researchers say in the paper describing their investigation of the role of inflammatory cytokine-induced lncRNAs in osteoarthritis pathogenesis and the underlying mechanisms, published in Science Signaling on May 24, 2022.

As a constituent of the noncoding "dark matter" of the genome, lncRNAs have important roles in regulating gene expression, by interacting directly with messenger RNA, but also with proteins, substrates and small molecules.

lncRNAs are increasingly implicated in multiple disease areas, and at the same time, are looked to as good drug targets, because they are expressed in a tissue-specific way.

Initial analysis of transcriptomics sequencing data of human knee osteoarthritic chondrocytes showed 1,041 previously annotated or potential lncRNAs were up- or downregulated by over 2-fold when the cells were stimulated with the inflammatory cytokines TNF-alpha and IL-1beta.

Of these, there was decreased expression of 718 lncRNAs, while 327 were highly upregulated in the inflamed cartilage cells.

Among these, the researchers, led by Su'an Tang, of the Clinical Research Center, Southern Medical University, Guandong, China, homed in on PILA, which was upregulated almost 15-fold. PILA consists of 3 exons with 554 nucleotides and is localized primarily to the nucleus of chondrocytes.

A real-time PCR analysis showed that expression of PILA quickly increased in response to TNF-alpha and IL-1beta stimulation. After peaking within 2 hours, levels gradually declined and returned to baseline after 12 hours.

It was shown that the number of PILA molecules per chondrocyte increased 12.5-fold, while in cartilage from patients undergoing knee replacement surgery, it was seen that PILA was increased in damaged, compared with undamaged tissue.

RNA interference

To investigate the mechanisms by which PILA plays a role in osteoarthritis pathology, the researchers carried out RNA interference experiments in human chondrocytes. Silencing PILA in TNF-alpha-treated chondrocytes reduced expression of matrix-degrading proteases such as MMP3, MMP13 and ADAMTS4, whereas overexpression of PILA promoted expression of these proteases.

Levels of the structural protein glycosaminoglycan were increased in PILA-silenced chondrocytes and reduced when PILA was overexpressed. It also was shown that knocking down PILA inhibited TNF-alpha-driven chondrocyte apoptosis. The reverse was the case when PILA was overexpressed.

NF-kappaB signaling is known to play a pivotal part in inflammatory osteoarthritis of the knee, and to establish how PILA fits into the picture, the researchers conducted an analysis of its spatial distribution, showing that PILA co-localizes with the p65 DNA binding subunit of NF-kappaB in osteoarthritic chondrocytes.

Cytokine-induced p65 phosphorylation and the subsequent translocation of p65 complexes into the nucleus is the hallmark of the activation of NF-kappaB signaling and the researchers found that silencing PILA decreased TNF-induced p65 phosphorylation, while overexpression promoted it. Immunofluorescence staining confirmed PILA knockdown abrogated TNF-induced p65 nuclear translocation.

"These findings demonstrate that highly abundant PILA promotes chondrocyte degradation and positively activates the NF-kappaB signaling pathway," say the researchers.

The finding that PILA expression was increased with TNF treatment prompted an investigation of the role of p65 in PILA transcription. It was found that silencing p65 significantly reduced PILA levels, suggesting transcription of the lncRNA depends on p65.

Sequence analysis pointed to a potential p65 binding site in the promoter region of PILA and this was confirmed experimentally, indicating p65 is primarily responsible for TNF-induced PILA transcription in chondrocytes.

That led on to further research to uncover the underlying mechanism that prompts PILA-induced activation of NF-kappaB signaling, and to the identification of PRMT1 (protein arginine N-methyltransferase 1) as the protein that binds PILA.

Curiously, although previous studies have reported that PRMT1 is increased in osteoarthritic chondrocytes, the researchers did not observe this, either in cells treated with TNF, or when comparing PRMT1 abundance in damaged and undamaged cartilage.

PILA abundance was not affected by PRMT1 knockdown, nor was PRMT1 abundance affected by PILA knockdown. However, PRTM1 is required for PILA to exert its effect on chondrocytes.

"These results suggest that PILA interacts with PRMT1 to form a functional complex that promotes chondrocyte dysfunction without regulating its abundance," the researchers say.

It has been reported that PRMT1 can bind to p65 and activate the NF-kappaB pathway, but again, the researchers did not observe this interaction. Rather, PRMT1 promoted methylation of the helicase DHX9, which in turn enhanced interaction of DHX9 with the NF-kappaB p65 subunit, promoting NK-kappaB pathway activation.

PILA was shown to promote the binding of PRMT1 to DHX9 in a concentration-dependent manner. That, in turn, modified DHX9, leading to increased expression of TGF-beta-activated kinase 1 (TAK1), which is the direct cause of NF-kappaB activation.

To investigate the physiological consequences of PILA activation, the researchers both knocked down and overexpressed the lncRNA in human cartilage and cultured it in the absence and presence of TNF for 10 days.

PILA overexpression promoted the loss of proteoglycans, even under noninflammatory conditions, whereas PILA knockdown alleviated TNF-induced degradation of the cartilage matrix.

In mice, delivery of PILA with a viral vector increased cartilage destruction.