New Results

Long non-coding RNA GRASLND enhances chondrogenesis via suppression of interferon type II signaling pathway

Nguyen P.T. Huynh, Catherine C. Gloss, Jeremiah Lorentz, Ruhang Tang, Jonathan M. Brunger, Audrey McAlinden, Bo Zhang, Farshid Guilak
doi: https://doi.org/10.1101/650010

Abstract

Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression and cellular processes; however, their roles in musculoskeletal development, disease, and regeneration remain poorly understood. Here, we identified a novel lncRNA, Glycosaminoglycan Regulatory ASsociated Long Non-coDing RNA (GRASLND) as a regulator of mesenchymal stem cell (MSC) chondrogenesis, and we investigated its basic molecular mechanism and its potential application towards regenerative medicine. GRASLND, a primate-specific lncRNA, is upregulated during MSC chondrogenesis and appears to act directly downstream of SRY-Box 9 (SOX9), but not Transforming Growth Factor Beta 3 (TGF-β3). Utilizing the established model of pellet formation for MSC chondrogenesis, we showed that the silencing of GRASLND resulted in lower accumulation of cartilage-like extracellular matrix, while GRASLND overexpression, either via transgene ectopic expression or by endogenous activation via CRISPR, significantly enhanced cartilage matrix production. GRASLND acts to inhibit interferon gamma (IFN-γ) by binding to Eukaryotic Initiation Factor-2 Kinase EIF2AK2. We further demonstrated that GRASLND exhibits a protective effect in engineered cartilage against interferon type II across different sources of chondroprogenitor cells. Our results indicate an important role of GRASLND in regulating stem cell chondrogenesis, as well as its therapeutic potential in the treatment of cartilage-related diseases, such as osteoarthritis.

Significance Long non-coding RNAs (lncRNAs) play critical roles in gene regulation and cellular physiology; however, the role of lncRNAs in controlling stem cell chondrogenesis remains to be determined. Here, we utilized next generation sequencing of adult stem cell chondrogenesis to identify a set of potential lncRNA candidates involved in this process. We identified lncRNA Glycosaminoglycan Regulatory ASsociated Long Non-coDing RNA (GRASLND) and characterized its molecular mechanism of action. We described a novel role of GRASLND in positive regulation of chondrogenesis via its inhibition of type II interferon. Importantly, we showed that overexpression of GRASLND augments stem cell chondrogenesis, providing a promising approach to enhancing stem cell chondrogenesis and cartilage regeneration.

Introduction

Articular cartilage is an aneural, avascular tissue and has little or no capacity for intrinsic repair (1). While there are currently no effective treatments available for cartilage repair, focal cartilage or osteochondral lesions generally progress to osteoarthritis (OA), a progressive degenerative disease characterized by changes in the articular cartilage and remodeling of other joint tissues such as the synovium and subchondral bone. Thus, there remains an important need for regenerative therapies that can enhance cartilage repair through tissue engineering or cell therapy approaches (2-7).

In this regard, adult stem cells such as bone marrow derived mesenchymal stem cells (MSCs) or adipose-derived stem cells (ASCs) provide a readily accessible source of multipotent cells that show significant promise for regenerative medicine (8–11). When cultured in a defined cocktail supplemented with Transforming Growth Factor Beta 3 (TGF-β3), MSCs produce a cartilaginous matrix that is rich in glycosaminoglycan (GAG) and collagen type II (COLII) (12, 13). However, the complete pathway involved in MSC chondrogenesis is not fully deciphered, and a detailed understanding of the gene regulatory networks that control this process could provide new insights that accelerate and improve cartilage regeneration from endogenous or exogenously grafted MSCs.

Increasing evidence suggests that such gene regulatory pathways operational in stem cell differentiation may rely not only on protein-coding RNAs, but also on non-coding RNAs (ncRNAs). Non-coding RNAs (ncRNAs) were initially difficult to identify because they neither possessed open reading frames, nor were they evolutionarily highly conserved (14). In one of the first landmark studies, chromatin-state mapping was used to identify transcriptional units of functional large intervening non-coding RNAs (lincRNAs) that were actively transcribed in regions flanking protein-coding loci (15), and follow-up loss-of-function studies indicated that these lincRNAs were indeed crucial for the maintenance of pluripotency in embryonic stem cells (16). There is a growing understanding of long non-coding RNA (lncRNA) function in a multitude of tissues and cellular processes. For example, detailed mechanistic studies on the role of lncRNAs in X chromosome inactivation (17), or nervous system development and functions (18, 19) have been previously reported. However, knowledge of their roles in the musculoskeletal system, particularly in chondrogenesis remains limited. Only a handful of functional studies have been carried out in this regard. For example, lncRNA-HIT (HOXA Transcript Induced by TGFβ) (20) has been shown to play a role in epigenetic regulation during early limb development. Other studies have implicated a specific lncRNA, ROCR (Regulator of Chondrogenesis RNA) (21), to act upstream of SRY-Box 9 (SOX9) and regulate chondrocyte differentiation (22).

As one of their many modes of actions, lncRNAs are also known to regulate and modulate various signaling cascades involved in the control of gene regulatory networks. Therefore, there may exist a connection between lncRNA candidates and signaling pathways previously known to play a role in the musculoskeletal system development. More specifically, there is growing evidence for the role of interferon (IFN) in skeletal tissue development and homeostasis (23–31). There are two main types of IFN. Type I includes mainly IFN alpha (IFN-α) and IFN beta (IFN-β) that form complexes with Interferon Alpha and Beta Receptors (IFNARs), activating the Janus Kinase/ Signal Transducers and Activators of Transcription (JAK/STAT) pathway by phosphorylation of STAT1 (Signal Transducer and Activator of Transcription 1) and STAT2 (Signal Transducer and Activator of Transcription 2). Phosphorylated STAT1/STAT2 then form complexes with IRF9 (IFN Regulatory Factor 9) and translocate into the nucleus to activate downstream targets via the interferon-stimulated responsible element (ISRE) DNA binding motif. Type II, on the other hand, relies on activation of the JAK/STAT pathway following the binding of IFN gamma (IFN-γ) to Interferon Gamma Receptors (IFNGRs). This subsequently results in the phosphorylation and dimerization of STAT1 that translocates into the nucleus and induces downstream targets via the gamma activated sequence (GAS) DNA binding element (32–34).

Although interferons (IFN) are widely known for their antiviral response, they can also act in other aspects of cellular regulation (33). Interestingly, IFN-γ has been implicated in non-viral processes, most notably its priming effect in auto-immune diseases such as lupus nephritis, multiple sclerosis, or rheumatoid arthritis (35). An additional goal of this study was to elucidate the link between IFN-γ and our lncRNA candidate, and how this interaction could potentially play a role in MSC chondrogenesis and cartilage tissue engineering.

In a recent publication, we used high-depth RNA sequencing to map the transcriptomic trajectory of MSC chondrogenesis (36). This dataset provides a unique opportunity to identify candidate genes for subsequent functional characterization as regulators of chondrogenesis. Here, we used bioinformatic approaches to integrate our RNA-seq data with other publicly available datasets, applying a rational and systematic data mining method to define a manageable list of final candidates for follow-up experiments. As a result, we identified RNF144A-AS1 to be a crucial regulator of chondrogenesis, and proposed the name Glycosaminoglycan Regulatory ASsociated Long Non-coDing RNA (GRASLND). We showed that GRASLND enhances chondrogenesis by acting to suppress the IFN-γ signaling pathway, and this effect was prevalent across different adult stem cell types and conditions. Together, these results highlight novel roles of GRASLND and its modulation of IFN in stem cell chondrogenesis, as well as its therapeutic potential to enhance cartilage regeneration.

Results

RNF144A-AS1 is crucial to and specifically upregulated in chondrogenesis

First, we utilized our published database on MSC chondrogenesis (GSE109503) (36) to identify long non-coding RNA candidates. We investigated the expression patterns of MSC markers (ALCAM, ENG, VCAM1), chondrogenic markers (ACAN, COL2A1, COMP), and SOX transcription factors (SOX5, SOX6, SOX9) (Figure S1A). Pearson correlation analysis revealed 141 lncRNAs whose expression was highly correlated to those of MSC markers, 40 lncRNAs to chondrogenic markers, and 17 lncRNAs to SOX transcription factors (Figure S1B, C). Among those, two were downregulated and two were upregulated upon ectopic SOX9 overexpression (Table 1) (GSE69110) (37). To validate their functions in chondrogenesis, we systematically designed small hairpin RNAs (shRNAs) targeting each candidate and assessed knockdown effect after 21 days of chondrogenic induction. We successfully designed two target shRNAs for each of three candidates, and one target shRNA for the other candidate (Figure S2). We showed that knockdown of two out of three MSC-related lncRNAs did not influence the production of glycosaminoglycans (GAG) - an important extracellular matrix component in cartilage (Figure S2). While these lncRNAs may have other regulatory functions in MSCs, their roles in chondrogenesis appeared to be minimal. Moreover, we found that lower levels of MSC-correlated lncRNAs did not prime the MSCs toward chondrogenesis. However, knockdown of RNF144A-AS1 (RNF144A Antisense RNA 1) resulted in decreased expression of chondrogenic markers (COL2A1, ACAN), and upregulation of apoptotic (CASP3) and cellular senescence (TP53) markers (Figure 1A, B). This effect was not due to nonspecific cytotoxicity of examined shRNAs, as released levels of lactase dehydrogenase (LDH) were similar among control and shRNA-expressing cells (Figure S3). In addition, biochemical assays indicated a reduction in GAG deposition (p < 0.0001) as well as DNA and GAG/DNA levels (p < 0.001) (Figure 1 C-E). Histologically, we observed the same phenotypic loss of GAG and collagen type II in the extracellular matrices (ECM) of pellet samples with RNF144A-AS1 targeted shRNAs, while the scrambled controls displayed explicit staining of these proteins (Figure 1F). Taken together, this data indicates that RNF144A-AS1 may be required for both cellular proliferation and cartilage-like matrix production.

Figure 1:
Figure 1:

RNF144A-AS1 is important and specifically upregulated in MSC chondrogenesis. (A) Expression pattern of RNF144A-AS1 in chondrogenesis (GSE109503 (36)). Log2TPM: log transformed value of transcripts per million (TPM). (B) Effect of RNF144A-AS1 knockdown on chondrogenic, apoptotic, and cell cycle inhibition markers (n = 5). (C-E) Effect of RNF144A-AS1 knockdown on pellet matrix synthesis (n = 5). (F) Representative histological images of day 21 MSC pellets. Scale bar = 200 µm. SafO-FG: SafraninO-Fast Green staining. COLII IHC: collagen type II immunohistochemistry. hOC: human osteochondral control. (G-I) qRT-PCR analysis of MSC samples cultured in (G) adipogenic condition (n=6), (H) osteogenic condition (n=6), (I) chondrogenic condition (n=3-4). One-way ANOVA followed by Tukey post-hoc test (α=0.05). Groups of different letters are statistically different from one another.

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Table 1:

Long non-coding RNA candidates shortlist

To establish whether RNF144A-AS1 expression is specific to chondrogenesis or involved in other differentiation pathways, MSCs were induced towards adipogenic, osteogenic, or chondrogenic lineages, and RNF144A-AS1 expression was measured at various timepoints throughout these processes. Successful differentiation was observed with an increase in lineage-specific markers: PPARG (Peroxisome Proliferator Activated Receptor Gamma) and ADIPOQ (Adiponectin, C1Q And Collagen Domain Containing) for adipogenesis, COL1A1 (Collagen Type I Alpha 1 Chain) and COL10A1 (Collagen Type X Alpha Chain 1) for osteogenesis, and ACAN (Aggrecan), SOX9 (SRY-Box 9) and COL2A1 (Collagen Type II Alpha Chain 1) for chondrogenesis (Figure 1 G-I). We found that RNF144A-AS1 expression was particularly enriched as chondrogenesis progressed (Figure 1I). In contrast, RNF144A-AS1 peaked at earlier timepoints during adipogenesis but decreased at later time points (Figure 1G), and downregulated when MSCs underwent osteogenic induction (Figure 1H), indicating that RNF144A-AS1 is specifically upregulated in chondrogenesis. Furthermore, we speculate that RNF144A-AS1 may display inhibitory effects on osteogenesis and adipogenesis, thus being downregulated during these processes.

To validate these gene expression findings, we performed RNA fluorescence in situ hybridization (FISH) throughout the time course of MSC chondrogenesis. Pellets exhibited RNF144A-AS1 FISH signals at later time points during chondrogenic differentiation, consistent with RNA-seq data (Figure 2A). Next, to confirm RNF144A-AS1 subcellular location, we performed qRT-PCR on isolated nuclear and cytoplasmic fractions of day 21 MSC pellets (Figure 2B). We compared the subcellular expression patterns of RNF144A-AS1 to NEAT1 (Nuclear Paraspeckle Assembly Transcript 1) and GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase). NEAT1 is a lncRNA previously characterized to localize to the nucleus (38, 39), and GAPDH is an mRNA and thus should be exported to the cytoplasm for protein synthesis. Consistent with previous findings, NEAT1 displayed lower expression in the cytoplasmic compared to the nuclear fraction, in contrast to GAPDH. By this measurement, RNF144A-AS1 exhibited higher expression in the cytoplasm, indicating its cytoplasmic subcellular location. Our finding was recapitulated by RNA in situ hybridization followed by confocal microscopy (Figure 2C). Interestingly, since RNF144A-AS1 showed punctate labeling, we speculate that this lncRNA may function in the form of an RNA-protein complex.

Figure 2:
Figure 2:

RNF144A-AS1 is localized to the cytoplasm. (A) RNA in situ hybridization of MSC-derived pellets at different time points during chondrogenesis. GAPDH and RNF144A-AS1 probes were hybridized on separate slides. Scale bar = 20 µm. (B) qRT-PCR of nuclear versus cytoplasmic fraction of day 21 MSC pellets (n=4). (C) Confocal microscopy on MSC-derived pellets. Scale bar = 5 µm. One-way ANOVA followed by Tukey post-hoc test (α=0.05). Groups of different letters are statistically different from one another.

Characterization of RNF144A-AS1

We examined the characteristics of RNF144A-AS1 by first exploring its evolutionary conservation. Except for exon 1, the genomic region of RNF144A-AS1 is highly conserved in primates (Homo sapiens, Pan troglodytes, and Rhesus macaque) whose common ancestor traced back to 25 million years ago (40), while sequences are less conserved in other mammals (Figure 3A). This suggests that RNF144A-AS1 may belong to a group of previously identified primate-specific lncRNAs (41, 42).

Figure 3:
Figure 3:

RNF144-AS1 relationship to RNF144A and SOX9. (A) RNF144A-AS1 genomic location and conservation across different species. Data retrieved from UCSC Genome Browser. (B) Knockdown of RNF144A-AS1 and expression of RNF144A (n=4). (C) Overexpression RNF144A-AS1 and expression of RNF144A (n=4). Welch’s t-test. (D) Protein amount of RNF144A by western blot in variation of RNF144A-AS1 levels. Lanes indicate biological replicates. (E) RNF144A-AS1 level in GFP- or SOX9-transduced MSCs under different doses of TGF-β3 (n=6). Two-way ANOVA followed by Tukey post-hoc test (α=0.05) on the effect of SOX9 overexpression (p < 0.0001) and doses of TGF-β3 (p > 0.05). The interaction between two tested factors (SOX9 overexpression and TGF-β3 doses) was not significant (p > 0.05). Groups of different letters are statistically different. ns: not significant.

Per GENCODE categorization, the AS (antisense) suffix indicates a group of lncRNAs that are positioned on the opposite strand, with overlapping sequences to their juxtaposed protein-coding genes. Often, these lncRNAs play a role in regulating the expression of their protein-coding counterparts (22). Therefore, we set out to examine whether this is also the case for RNF144A-AS1 (Figure 3B-C). Neither knockdown nor overexpression of RNF144A-AS1 affected RNF144A transcript levels in MSCs cultured with or without TGF-β3. Moreover, RNF144A protein translation also remained unaffected with variations of RNF144A-AS1 levels, as indicated by western blot (Figure 3D). These results indicate that RNF144A-AS1 is not involved in the regulation of RNF144A. For these reasons, we proposed an alternative name for RNF144A-AS1: GRASLND - Glycosaminoglycan Regulatory ASsociated Long Non-coDing RNA.

Next, we explored the signaling axis of GRASLND. Data mining and computational analysis on earlier published data suggested that GRASLND was a downstream effector of SOX9 (GSE69110) (37). When SOX9 was overexpressed in fibroblasts, GRASLND expression was increased (∼ 2-fold). We further confirmed this by utilizing SOX9 transgene overexpression in our MSCs culture (Figure 3E). Interestingly, while TGF-β3 has been demonstrated to act upstream of SOX9, exogenous addition of this growth factor alone did not result in enhanced GRASLND expression. It is notable that SOX9 levels in GFP controls were indistinguishable between TGF-β3 conditions at the time of investigation (1 week in monolayer culture), consistent with our previous finding that SOX9 was not upregulated until later timepoints in MSC chondrogenesis (36). Therefore, TGF-β3, despite being a potent growth factor, is not sufficient to elevate GRASLND expression. Instead, GRASLND appeared to be a downstream target of SOX9.

Enhanced chondrogenesis for cartilage tissue engineering with GRASLND

As knockdown of GRASLND inhibited GAG and collagen deposition, we sought to investigate whether overexpression of GRASLND would enhance chondrogenesis. We assessed this question by both transgene ectopic expression and by CRISPR-dCas9 (Clustered regularly interspaced short palindromic repeats – catalytically dead Cas9) mediated in-locus activation.

We designed our lentiviral transfer vector to carry a BGH-pA (Bovine Growth Hormone Polyadenylation) termination signal downstream of GRASLND to allow for its correct processing (Figure S4A). Additionally, GRASLND was also driven under a doxycycline inducible promoter, enabling the temporal control of its expression. We utilized this feature to induce GRASLND only during chondrogenic culture (Figure 4A). This experimental design focused solely on the role of GRASLND during chondrogenesis, while successfully eliminating its effect in MSC maintenance and expansion from our analysis. As control, a vector encoding the Discosoma sp. red fluorescent protein (dsRed) coding sequence in place of GRASLND was utilized. Since doxycycline was most potent at 1 µg/mL (Figure S4 B-C), this dose was used for all following experiments.

Figure 4:
Figure 4:

GRASLND enhances chondrogenesis. (A) Experimental timeline. (B,C) Biochemical analyses of day 21 MSC pellets (n=4). Welch’s t-test. (D,E) qRT-PCR analyses of day 21 MSC pellets (n=5 in D; n=6 in E). Welch’s t-test. (F) Representative histological images of day 21 MSC pellets. COLII IHC: collagen type II immunohistochemistry. hOC: human osteochondral control. Scale bar = 100 µm. (B,D,F) Transgene ectopic expression of GRASLND. (C,E,F) CRISPR-dCas9-VP64 induced activation of GRASLND. ns: not significant (p > 0.05).

To determine whether GRASLND would improve chondrogenesis at lower doses of growth factor or at earlier time points, we compared DNA and GAG levels from pellets cultured under different TGF-β3 concentrations on day 7, day 14, and day 21 (Figure S4 D-F). In agreement with our knockdown data, DNA content was unaffected. On the other hand, increases in GAG were observed at higher doses and at later time points, especially at 10 ng/mL of TGF-β3. It appears that an elevated level of GRASLND alone was not sufficient to enhance GAG deposition, and GRASLND may act in concert with other downstream effectors, which were not present at lower doses of TGF-β3 or at earlier time points in the process.

Elevated levels of GRASLND resulted in higher amounts of GAG deposition (p < 0.001) (Figure 4B), consistent with our data on the gene expression level (Figure 4D). We observed a slight increase in chondrogenic markers (COL2A1, ACAN), and a slight decrease in the apoptotic marker CASP3, while cellular senescence was not different between the two groups (TP53) (Figure 4D). Histologically, pellets derived from dsRed-transduced MSCs exhibited normal GAG and collagen type II staining, indicating successful chondrogenesis. The control pellets were indistinguishable from those derived from GRASLND-transduced MSCs (Figure 4F), albeit macroscopically smaller at the time of harvest.

These findings were further confirmed using CRISPR-dCas9-VP64 mediated activation of endogenous GRASLND. This system had been previously utilized to upregulate various transcription factors that efficiently induce embryonic fibroblasts into neurons (43, 44). After screening eleven synthetic gRNAs, we selected the one with highest activation level (Figure S5). When GRASLND was transcriptionally activated with CRISPR-dCas9, chondrogenesis was enhanced as evidenced by elevated amount of GAG deposition (p < 0.01); DNA amount may also be slightly increased, albeit not statistically significant (Figure 4C). Similar trends were detected by qRT-PCR (Figure 4E) and histology (Figure 4F). It is worth noting that CRISPR-dCas9 mediated activation only resulted in a moderate up-regulation of GRASLND relative to transgene ectopic expression (2-fold vs 100-fold). However, the functional outcome was more pronounced with CRISPR-dCas9. We observed approximately 50% increase in the level of GAG produced when normalized to DNA (9.4 ± 2.19 mg/mg vs 16.3 ± 2.08 mg/mg), compared to 30% detected with ectopic expression (10.5 ± 0.84 mg/mg vs 13.9 ± 0.52 mg/mg).

GRASLND inhibits type II interferon signaling potentially by binding to EIF2AK2 and protects engineered cartilage from interferon

To decipher the potential signaling pathways involved, we chondrogenically induced MSCs in the presence or absence of GRASLND, and then utilized RNA-seq to compare the global transcriptomic changes between two conditions. As expected, GRASLND depletion resulted in impaired expression of chondrocyte-associated genes such as TRPV4 and COL9A2 (top 20 downregulated genes ranked by adjusted p-values) (Figure 5A). Skeletal system development and extracellular matrix organization were among the pathways most affected by the knockdown (Figure 5B). Surprisingly, pathways pertaining to interferon response were highly enriched in the upregulated gene list upon silencing of GRASLND. The top 20 upregulated genes involved many IFN downstream targets (MX2, IFI44, IFI44L, IFITM1, IFI6, IFIT1, STAT1, MX1, IFIT3, OAS3, OAS2), with both type I (IFN-α, IFN-β) and type II (IFN-γ) found to be enriched in our gene ontology analysis (Figure 5B). Furthermore, upregulated genes were also found to exhibit DNA binding motifs for transcription factors of the IFN pathways: STAT1, STAT2, IRF1, IRF2 (Table 2). A full list of differentially expressed genes is provided in Supplementary Materials. Further bioinformatic analyses created a network of potential transcription regulators as well as gene ontology terms for the upregulated gene cohort as a result of GRASLND silencing (Figure 5C). Taken together, GRASLND may potentially act to suppress the activities of these transcription factors, as a result affecting IFN signaling pathways during chondrogenesis.

Figure 5:
Figure 5:

GRASLND suppresses interferon type II signaling. (A) Top 20 up- and down-regulated genes in GRASLND KD pellets compared to scrambled controls. (B) Gene ontology analysis of affected pathways. (C) Upregulated targets and related gene ontology terms and potential transcription factors. (D,E) Luciferase reporter assays on MSCs transduced with: (D) ISRE promoter element (n=3), or (E) GAS promoter element (n=3). Two-way ANOVA followed by Tukey post-hoc test (α=0.05). Groups of different letters are statistically different. (F) Biochemical assays on MSC-derived pellets cultured under 100 ng/mL of IFN-β (n=4). (G) Biochemical assays on MSC-derived pellets cultured under 5 ng/mL of IFN-γ (n=6). Welch’s t-test. ns: not significant.

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Table 2:

Top 5 enriched Cis-BP motifs and associated transcription factors for upregulated genes upon GRASLND knockdown

To further confirm this relationship, we performed luciferase reporter assays for interferon signaling upon GRASLND knockdown. Utilizing specific reporter constructs, we were able to determine whether GRASLND acted on type I or type II IFN. Our results indicated that decreased level of GRASLND led to heightened type II (IFN-γ) (Figure 5E) response but not type I (IFN-β) (Figure 5D). Importantly, luminescence activities between scrambled control and GRASLND knockdown were indistinguishable from each other in basal, IFN-free conditions. This indicates that at basal level, the two groups responded similarly to lentiviral transduction, and the observed difference in IFN signal was a consequence of GRASLND downregulation.

Since GRASLND was expressed in the cytoplasm (Figure 2C), we hypothesized that it is part of an RNA-protein complex. To test this, we performed an RNA pull down assay, followed by mass spectrometry. Here, streptavidin beads were used as control, or conjugated to sense or antisense strands of GRASLND. Naked or conjugated beads were then incubated with lysates from day 21 pellets, from which bound proteins were eluted for further analyses. We found that Interferon-Induced Double-Stranded RNA-Activated Protein Kinase (EIF2AK2) peptides were detected at elevated levels in sense samples as compared to antisense controls (p < 0.05); peptides were undetected in naked bead controls. Subsequent RNA pull-down followed by western blot confirmed EIF2AK2 as a binding partner of GRASLND (Figure S6). We detected an increased level of EIF2AK2 bound to the sense strand of GRASLND relative to the antisense or the pellet lysate control. We speculate that this association of GRASLND RNA to EIF2AK2 could potentially result in downregulation of IFN-γ signaling.

Interestingly, by mining a published microarray database (GSE57218) (45), we found that IFN-related genes were highly elevated in cartilage tissues of osteoarthritis patients: STAT1, IFNGR2, NCAM1, MID1 (Figure S7A). Since the microarray did not contain probes for GRASLND, no information on its expression could be extracted. In addition, we identified another independent study that reported changes in the transcriptomes of intact and damaged cartilage tissues (E-MTAB-4304) (46). Similarly, a cohort of IFN-related genes were also upregulated in damaged cartilage, especially STAT1 and IFNGR1 (Figure S7B). Interestingly, we identified a negative correlation between GRASLND and a few IFN related genes (IFNGR1, ICAM1) in damaged cartilage (Figure S7C). Therefore, we proposed that GRASLND may possess some therapeutic potential through suppressing IFN signaling in osteoarthritis. To evaluate this possibility, we implemented the use of the GRASLND transgene in engineered cartilage cultured under IFN addition (100 ng/mL of IFN-β or 5 ng/mL of IFN-γ). We determined doses of IFN-β and IFN-γ by selecting the lowest concentration at which day 21 pellets exhibited GAG loss compared to no IFN control. Consistent with luciferase reporter assays, the protective effect of GRASLND was observed upon IFN-γ challenge but not IFN-β (Figure 5F, G). However, we observed a reduced level of GAG production compared to normal conditions, suggesting that GRASLND can protect the ECM from degradation, but not completely to control levels.

GRASLND enhanced the chondrogenesis of adipose-derived stem cells

To determine if the function of GRASLND is unique to MSCs or present in other adult stem cells, we addressed whether modulating GRASLND expression could also improve chondrogenesis of adipose stem cells (ASCs). We observed an increase in GAG production when GRASLND was overexpressed in ASCs compared to control (p < 0.0001) (Figure 6A), although ACAN levels were not significantly increased. Importantly, COL2A1 expression was significantly elevated (∼ 5-fold) with overexpression of GRASLND (Figure 6B). Histologic examination of the engineered cartilage showed a similar level of collagen type II in pellets with GRASLND overexpression compared to the dsRed control (Figure 6C). Based on these data, it appears that GRASLND utilized the same mechanism across these two cell types, asserting a pan effect on potentiating their chondrogenic capabilities.

Figure 6:
Figure 6:

GRASLND enhances chondrogenesis in adipose-derived stem cells. (A) Biochemical analyses (n= 5). (B) qRT-PCR analyses (n=6). (C) Representative histological images of day 21 ASC pellets. COLII IHC: Collagen type II immunohistochemistry. hOC: Human osteochondral control. Scale bar = 100 µm. Welch’s t-test. ns: not significant.

Discussion

Here, we identified and demonstrated the first functional study of lncRNA GRASLND, which acts to enhance stem cell chondrogenesis. Knockdown of GRASLND via shRNA inhibited chondrogenesis, whereas ectopic transgene or CRISPR-based overexpression of GRASLND enhanced chondrogenesis of MSCs and ASCs. Pathway analysis revealed a link between GRASLND and IFN-γ signaling pathway in this process, which was confirmed by the identification of EIF2AK2 as its binding partner. Unfortunately, lack of a known murine homolog makes it difficult to study GRASLND in vivo, and thus future studies may require GRASLND transgenic models in primate species.

In the context of the musculoskeletal system, IFN is mostly recognized for its role in bone development and homeostasis (23–27, 30), myogenesis (29, 47, 48), as well as its crosstalk with TGF-β in wound healing (49). Notably, IFN-γ has been suggested to inhibit collagen synthesis in dermal fibroblasts, myofibroblasts, and articular chondrocytes (49–53). Furthermore, the JAK/STAT pathway, which involves IFN downstream effectors, has also been shown to inhibit chondrocyte proliferation and differentiation (28, 31). Here, we found that GRASLND acts to suppress the IFN mechanism. In addition, we also present evidence indicating an interaction between GRASLND and EIF2AK2 (also referred to as PKR). Canonically a crucial player in protein synthesis, PKR has also been reported to control STAT signaling by directly binding to and preventing its association with DNA for gene activation (54, 55). Additionally, several studies have suggested that highly structured, single stranded RNA can also activate PKR via its double stranded RNA binding domains (dsDRBs) (56–60). Our RNA-seq data suggested that upon GRASLND knockdown, a cohort of downstream targets of STATs were upregulated.

Based on the presence of DNA binding motifs in investigated targets, we identified both STAT1 and STAT2 as potential regulators of genes disrupted by GRASLND knockdown. However, our luciferase reporter assays pointed towards a mechanism in IFN type II (gene activation by STAT1 homodimer) rather than type I (gene activation by STAT1/STAT2 heterodimer). Thus, we hypothesized that GRASLND could form a secondary structure to bind and activate PKR, which in turn inhibits STAT1-related transcriptional function. This mechanism supports the hypothesis that modulation of IFN-γ via the JAK/STAT pathway, achieved by the GRASLND/PKR RNA-protein complex, is important for cellular proliferation and differentiation during chondrogenesis.

Upregulation of IFN has also been implicated in arthritis by several studies (61–64). Publicly available databases provide evidence corroborating similar patterns of IFN in degenerated cartilage. As GRASLND inhibits IFN, utilization of this lncRNA offers potential in both MSC cartilage tissue engineering and in OA treatment. As a proof of concept, we showed that GRASLND could enhance matrix deposition across cell types of origin, with and without interferon challenge in vitro. It would be interesting to next investigate whether GRASLND can protect cartilage from degradation in a milieu of pro-inflammatory cytokines in vivo.

Since lentivirus was employed to manipulate the expression of GRASLND, it is possible our observations were confounded by the cellular response to viral infection. However, our luciferase reporter assays demonstrated that basal luminescence levels (with no interferon supplementation) between the scrambled controls and the shRNA treatments were indistinguishable. This suggests that altered levels of interferon signaling can be attributed to experimentally varied levels of GRASLND and not to the presence of lentivirus. Our data indicate that GRASLND acts through type II rather than type I IFN. We found that 5 ng/mL of IFN-γ was still more detrimental to chondrogenic constructs compared to 100 ng/mL of IFN-β. One potential explanation for this phenomenon may be the skewed distribution of available surface receptors between type I and type II (IFNAR vs IFNGR). Indeed, MSCs express a much lower level of IFNAR2 compared to IFNAR1, IFNGR1, or IFNGR2 (both in GSE109503 (36) and in GSE129985 (this manuscript)). As these receptors function as heterodimers (32, 34), response to type I may be stunted due to IFNAR2 deficiency.

Furthermore, we showed that a modified CRISPR-dCas9 system could successfully be used for endogenous transcriptional activation of lncRNA. This system had been previously used in other cell types to regulate expression of both protein-coding and non-coding genes (43, 44, 65, 66). We showed that CRISPR may be more effective than transgene expression, as indicated by a larger increase in GAG production, despite lower levels of overall gene activation. As GRASLND does not regulate RNF144A, it is evident that GRASLND acts in trans. However, we speculate the CRISPR-dCas9 system could also be useful for gain of function studies to investigate lncRNAs acting in cis, as well as lncRNAs that are difficult to obtain via molecular cloning due to their secondary structures, high repeated sequence or GC-rich content.

In conclusion, we have identified GRASLND as an important regulator of chondrogenesis. GRASLND acts downstream of SOX9 and enhances cartilage-like matrix deposition in stem cell-derived constructs. Moreover, GRASLND functions to suppress IFN via PKR, and as a result induces adult stem cells towards a more chondrocyte-like lineage. It is likely that the GRASLND/PKR RNA-protein complex may inhibit STAT1 transcriptional activity. We propose that GRASLND can potentially be applied therapeutically for both cartilage tissue engineering and for the treatment of OA.

Materials and Methods

Cell culture

Bone marrow was obtained from discarded and de-identified waste tissue from adult bone marrow transplant donors in accordance with the Institutional Review Board of Duke University Medical Center. Adherent cells were expanded and maintained in expansion medium: DMEM-low glucose (Gibco), 1% Penicillin/streptomycin (Gibco), 10% fetal bovine serum (FBS) (ThermoFisher), and 1 ng/mL basic fibroblast growth factor (Roche) (67).

Adipose derived stem cells (ASCs) were purchased from ATCC (SCRC-4000) and cultured in complete growth medium: Mesenchymal stem cells basal medium (ATCC PCS-500-030), mesenchymal stem cell growth kit (ATCC PCS-500-040) (2% FBS, 5 ng/mL basic recombinant human FGF, 5 ng/mL acidic recombinant human FGF, 5 ng/mL recombinant human EGF, 2.4 nM L-alanyl-L-glutamine), 0.2 mg/mL G418.

Plasmid construction

shRNA

Short hairpin RNA (shRNA) sequences for specific genes of interest were designed with the Broad Institute GPP Web Portal (68). For each gene, six different sequences were selected for screening, after which the two most effective were chosen for downstream experiments in chondrogenic assays. Selected shRNAs were cloned into a modified lentiviral vector (Addgene #12247) using MluI and ClaI restriction sites, as described previously (69). A complete list of effective shRNA sequences is presented in Table S1.

Transgene overexpression of GRASLND

A derivative vector from modified TMPrtTA (3, 70) was created with NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs). Backbone was digested with EcoRV-HF (New England Biolabs) and PspXI (New England Biolabs). The following resultant fragments were amplified by polymerase chain reaction and assembled into the digested plasmid: Tetracycline responsive element and minimal CMV promoter (TRE/CMV), Firefly luciferase, bGH poly(A) termination signal (BGHpA). Primers and plasmids for cloning are provided in Table S2.

The full sequence of GRASLND transcript variant 1 (RefSeq NR_033997.1) was synthesized by Integrated DNA Technologies, Inc. GRASLND or the Discosoma sp. red fluorescent protein coding sequence (dsRed) were cloned into the above derivative tetracycline inducible plasmid with NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) at NheI and MluI restriction sites (pLVD-GRASLND and pLVD-dsRed). Amplifying primers are provided in Table S2.

CRISPR-dCas9 activation of GRASLND

Guide RNA sequences were designed using the UCSC genome browser (http://genome.ucsc.edu/) (71), integrated with the MIT specificity score calculated by CRISPOR and the Doench efficiency score (72, 73). Oligonucleotides (IDT, Inc) were phosphorylated, annealed, and ligated into the pLV-hUbC-dCas9-VP64 lentiviral transfer vector (Addgene #53192) previously digested at BsmBI restriction sites (74). Eleven potential guide RNA sequences were selected and screened for their efficacy, and the gRNA with the highest activation potential was chosen for further experiments (Figure S5). The synthetic gRNA used in all CRISPR-dCas9 activation experiments has the following sequence: 5′-CCACTGGGGATAGTTCCCTG-3′.

Chondrogenesis assay

MSCs or ASCs were digested in 0.05% Trypsin-EDTA (Gibco), and trypsin was inactivated with 1.5X volume of expansion medium. Dissociated cells were centrifuged at 200 × g for 5 minutes, and supernatant was aspirated. Subsequently, cells were washed in pre-warmed DMEM-high glucose (Gibco) three times, and resuspended at 5 × 105 cells/mL in complete chondrogenic medium: DMEM-high glucose (Gibco), 1% Penicillin/ streptomycin (Gibco), 1% ITS+ (Corning), 100 nM Dexamethasone (Sigma-Aldrich), 50 µg/mL ascorbic acid (Sigma-Aldrich), 40 µg/mL L-proline (Sigma-Aldrich), 10 ng/mL rhTGF-β3 (R&D Systems). Five hundred µL of the above cell mixture was dispensed into 15 mL conical tubes, and centrifuged at 200 × g for 5 minutes. Pellets were cultured at 37°C, 5% CO2 for 21 days with medium exchange every three days.

Osteogenesis and adipogenesis assays

MSCs were plated at 2 × 104 cells/well in 6-well plates (Corning) and cultured for 4 days in MSC expansion medium, followed by induction medium for 7 days. Osteogenic induction medium includes: DMEM-high glucose (Gibco), 10% FBS, 1% Penicillin/ streptomycin (Gibco), 10 nM Dexamethasone (Sigma-Aldrich), 50 µg/mL ascorbic acid (Sigma-Aldrich), 40 µg/mL L-proline (Sigma-Aldrich), 10 mM β-glycerol phosphate (Chem-Impex International), 100 ng/mL rh-BMP2 (ThermoFisher). Adipogenic induction medium includes: DMEM-high glucose (Gibco), 10% FBS (ThermoFisher), 1% Penicillin/ streptomycin (Gibco), 1% ITS+ (Corning), 100 nM Dexamethasone (Sigma-Aldrich), 450 µM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 200 µM indomethacin (Sigma-Aldrich).

Biochemical assays

Harvested pellets were stored at −20°C until further processing. Collected samples were digested in 125 µg/mL papain at 60°C overnight. DMMB assay was performed as previously described to measure GAG production (76). PicoGreen assay (ThermoFisher) was performed to measure DNA content following manufacture’s protocol.

Immunohistochemistry and histology

Harvested pellets were fixed in 4% paraformaldehyde for 48 hours, and processed for paraffin embedding. Samples were sectioned at 10 µm thickness, and subjected to either Safranin O – Fast Green standard staining (77) or to immunohistochemistry of collagen type II (Developmental Studies Hybridoma Bank, University of Iowa; #II-II6B3). Human osteochondral sections were stained simultaneously to serve as positive control. Sections with no primary antibodies were used as negative control for immunohistochemistry.

RNA fluorescence in situ hybridization (RNA FISH)

Harvested pellets were snap frozen in Tissue-Plus O.C.T. Compound (Fisher HealthCare) and stored at −80°C until further processing. Samples were sectioned at 5 µm thickness and slides were stored at −80°C until staining. Probe sets for RNA FISH were conjugated with Quasar® 670 dye, and were synthesized by LGC Biosearch Technologies and listed in Table S3 (RNF144A-AS1). GAPDH probe set was pre-designed by the manufacturer. Staining was carried out according to manufacturer’s protocol for frozen tissues. Slides were mounted with Prolong Gold anti-fade mountant with DAPI (ThermoFisher) and imaged with the Virtual Slide Microscope VS120 (Olympus) at lower magnification and with the confocal microscope (Zeiss) at higher magnification.

RNA isolation and quantitative RT-PCR

Norgen Total RNA Isolation Plus Micro Kits (Norgen Biotek) were used to extract RNA from pellet samples and Norgen Total RNA Isolation Plus Kits (Norgen Biotek) were used for all other RNA isolation. For monolayer, cells were lysed in buffer RL and stored at −20°C until further processing. For pellets, harvested samples were snap frozen in liquid nitrogen and stored at −80°C until further processing. On day of RNA isolation, pellets were homogenized in buffer RL using a bead beater (BioSpec Products) at 2,500 oscillations per minute for 20 seconds for a total of three times. Subsequent steps were performed following manufacturer’s protocol.

Nuclear and cytoplasmic fractions from day 21 MSC pellets were separated with the NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher) following manufacturer’s protocol. Resulting extracts were immediately subjected to RNA isolation using the Norgen Total RNA Isolation Plus Micro Kits (Norgen Biotek) by adding 2.5 parts of buffer RL to 1 part of extract. Subsequent steps were carried out following manufacturer’s protocol.

Reverse transcription by Superscript VILO cDNA master mix (Invitrogen) was performed immediately following RNA isolation. cDNA was stored at −20°C until further processing. qRT-PCR was carried out using Fast SyBR Green master mix (Applied Biosystems) following manufacturer’s protocol. A complete list of primer pairs (synthesized by Integrated DNA Technologies, Inc.) is reported in Table S4.

Luminescence assay

MSCs were plated at 8.5 × 104 cells per well in 24-well plates (Corning). Lentivirus carrying the response elements for type I (ISRE - #CLS-008L-1) or type II (GAS - #CLS-009L-1) upstream of firefly luciferase was purchased from Qiagen. Twenty-four hours post plating, cells were co-transduced with virus in the following groups: ISRE with scrambled shRNA, ISRE with GRASLND shRNA, GAS with scrambled shRNA, GAS with GRASLND shRNA. Twenty-four hours post-transduction, cells were rinsed once in PBS and fresh medium was exchanged.

Three days later, medium was switched to expansion medium with 100 ng/mL IFN-β (PeproTech) for wells with ISRE or with 5 ng/mL IFN-γ (PeproTech) for wells with GAS. MSCs were cultured for another 22 hours, and then harvested for luminescence assay using Bright-Glo Luciferase Assay System (Promega). Luminescence signals were measured using the Cytation 5 Plate reader (BioTek).

Western blot

On day of harvest, cells were homogenized with complete lysis buffer in ice cold PBS: 10X RIPA buffer (Cell Signaling Technology), 100X phosphatase inhibitor cocktail A (Santa Cruz Biotechnology), 100X HaltTM protease inhibitor cocktails (ThermoScientific). Lysates were subsequently centrifuged at 14,000 × g for 15 minutes at 4°C, and supernatants were collected and stored at −20°C until further processing. Western blot was serviced by RayBiotech with the following antibodies: primary anti-β-actin (RayBiotech), primary anti-RNF144A (Abcam), primary anti-PKR (RayBiotech) and secondary anti-rabbit-HRP (horse radish peroxidase) (RayBiotech).

Statistical analyses

All statistical analyses were performed using R (78). Results from biochemical assays are depicted as mean ± SD. Results from qRT-PCR are depicted as fold-change with error bars calculated per Applied Biosystems manual instruction.

Additional methods are provided in supplemental information.

Author contributions

N.P.T.H, F.G designed research; N.P.T.H., C.C.G., J.L. and R.T. performed research and analyzed data; J.M.B., A.M., and B.Z. provided critical discussion and comments. N.P.T.H. and F.G. wrote the manuscript; all authors edited the manuscript.

Accession number: GSE129985

Acknowledgments

We thank the Genome Technology Access Center at Washington University in St Louis, the Proteomics Core Laboratory, and the Hope Center Viral Vectors Core for their resources and support. The CRISPR-dCas9-VP64 system was a generous gift from Dr. Charles Gersbach. We also wish to thank Sara Oswald for providing assistance in technical writing of the manuscript. This work was supported by the Arthritis Foundation, NIH grants AR50245, AR48852, AG15768, AR48182, AR067467, AR057235, AR073752, the Nancy Taylor Foundation for Chronic Diseases, and the Collaborative Research Center of the AO Foundation, Davos, Switzerland.

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Posted May 24, 2019.
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Supplementary Material

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Long non-coding RNA GRASLND enhances chondrogenesis via suppression of interferon type II signaling pathway
Nguyen P.T. Huynh, Catherine C. Gloss, Jeremiah Lorentz, Ruhang Tang, Jonathan M. Brunger, Audrey McAlinden, Bo Zhang, Farshid Guilak
bioRxiv 650010; doi: https://doi.org/10.1101/650010
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