Reduced glycolysis links resting zone chondrocyte proliferation in the growth plate

A gain-of-function mutation of the chondrocyte-specific microRNA, miR-140-5p, encoded by the MIR140 gene, causes spondyloepiphyseal dysplasia, Nishimura type (SEDN, also known as SED, MIR140 type; MIM, 611894). We reported that a mouse model for SEDN showed a unique growth plate phenotype that is characterized by an expansion of the resting zone of the growth plate and an increase in resting chondrocytes, of which the mechanism of regulation is poorly understood. We found that the miR-140 mutant chondrocytes showed a significant reduction of Hif1a, the master transcription factor that regulates energy metabolism in response to hypoxia. Based on this finding, we hypothesized that energy metabolism plays a regulatory role in resting chondrocyte proliferation and growth plate development. In this study, we show that suppression of glycolysis via LDH ablation causes an expansion of the resting zone and skeletal developmental defects. We have also found that reduced glycolysis results in reduced histone acetylation in the miR-140 mutant as well as LDH-deficient chondrocytes likely due to the reduction in acetyl-CoA generated from mitochondria-derived citrate. Reduction in acetyl-CoA conversion from citrate by deleting Acly caused an expansion of the resting zone and a similar gross phenotype to LDH-deficient bones without inducing energy deficiency, suggesting that the reduced acetyl-CoA, but not the ATP synthesis deficit, is responsible for the increase in resting zone chondrocytes. Comparison of the transcriptome between LDH-deficient and Acly-deficient chondrocytes also showed overlapping changes including upregulation in Fgfr3. We also confirmed that overexpression of an activation mutation of Ffgr3 causes an expansion of resting zone chondrocytes. These data demonstrate the association between reduced glycolysis and an expansion of the resting zone and suggest that it is caused by acetyl-CoA deficiency, but not energy deficiency, possibly through epigenetic upregulation of FGFR3 signaling.


Introduction
Skeletal development requires tightly coordinated proliferation and differentiation of growth plate chondrocytes, and aberrations of this process lead to diverse types of skeletal dysplasia 1 . In the growth plate, chondrocytes in the resting zone slowly proliferate, differentiate into vigorously proliferating columnar chondrocytes, then further into hypertrophic chondrocytes which drive longitudinal bone growth 2 . The resting zone chondrocytes are the most immature chondrocytes in the growth plate and are considered to serve as tissue-specific stem/progenitor cells; however, limited knowledge is available regarding the mechanisms by which the proliferation and differentiation of resting chondrocytes are regulated.
We previously reported that a mouse model for spondyloepiphyseal dysplasia, Nishimura type (SEDN, also known as SED, MIR140 type; MIM, 611894), caused by a gain-of-function mutation of microRNA-140-5p (miR-140-5p) that is encoded by the MIR140 gene, show a unique growth plate phenotype where the growth plate is expanded mainly due to the increase in the number of resting zone chondrocytes 3 . The RNA-seq data suggested reduced hypoxic adaptation in the mutant chondrocytes, including the reduced expression of the master transcription factor, hypoxia-inducible factor alpha (Hif1a). Since a binding site of mutant miR-140 was found in the coding sequence of Hif1a, it was suggested that the mutant miR-140-5p directly suppresses Hif1a expression. Hif1a is an essential transcription factor for cell survival and proliferation of growth plate chondrocytes 4; 5 . In hypoxic environments, like in developing growth plates, Hif1a protein is stabilized, and it stimulates glycolysis and suppresses mitochondrial metabolism by inhibiting pyruvate entry to mitochondria 6 . Live cells actively metabolize diverse molecules to meet their demands for energy and biomolecule synthesis. At the same time, various metabolites produced during energy metabolism are known to have regulatory roles in diverse cell functions 7; 8 . Therefore, in addition to energy deficiency, it is also possible that the altered metabolic state of the mutant miR-140-5p-expressing chondrocytes causes the deregulation of signaling pathways and gene expression critical for controlling chondrocyte proliferation and differentiation.
Adenosine triphosphate (ATP), cells' energy currency, is generated primarily by glucose catabolism. During glycolysis, a single molecule of glucose is broken down into pyruvate, while generating 2 molecules of ATPs. Pyruvate is further transported into mitochondria and metabolized into acetyl CoA in the tricarboxylic acid (TCA) cycle to generate electron donors. The electron transport chain then creates proton gradients, which drive ATP synthase to efficiently generate ATPs while consuming oxygen, through the process called oxidative phosphorylation (OXPHOS). Pyruvate can be directly metabolized in the cytoplasm into lactic acid by the enzyme, lactate dehydrogenase (LDH), which is encoded by the Ldha, Ldhb, Ldhc, and Ldhd genes in mice. This process is considered to be necessary to produce nicotinamide adenine dinucleotide (NAD + ), which is an essential co-substrate for glyceraldehyde-3-phosphate dehydrogenase (Gapdh), a key enzyme for glycolysis 9 . Therefore, by reducing LDH activity, glycolysis can be suppressed at an upstream step of the glycolytic cascade.
The growth plate is an avascular tissue, and therefore, it is mostly under a hypoxic condition, particularly during development. With the limited availability of oxygen, chondrocytes primarily rely on glycolysis for energy production 10 . This notion is supported by the fact that inhibition of glucose uptake in chondrocytes resulted in reduced chondrocyte proliferation and developmental defects of endochondral bones 11 . Although glycolysis is the major mechanism for ATP production in chondrocytes, mitochondrial metabolism still plays an important role in growth plate chondrocytes, as conditional deletion of Tfam, a transcription factor critical for mitochondrial genome transcription, impairs normal endochondral bone development 12 . In addition to energy production, mitochondria play important regulatory roles by providing various bioactive molecules. For example, citrate in the TCA cycle is exported to the cytoplasm and converted back into acetyl-CoA, which is the primary source for lipid synthesis as well as the donor of the acetyl group for protein modification 7; 8 . It is known that Ac-CoA availability directly determines the acetylation level of proteins, including histones, the most abundantly acetylated group of proteins in the cell 13 .
Given the importance of glucose metabolism in chondrocytes and the unique growth plate phenotype of the SEDN mouse model that shows reduced Hif1a expression and altered metabolic gene expression, we hypothesize that energy metabolism has regulatory roles in growth plate development.
In this study, we deleted the major LDH genes, Ldha and ldhb to suppress glycolysis in chondrocytes. We show that LDH ablation causes skeletal developmental defects with increased resting chondrocyte proliferation. LDH deficiency causes compensatory upregulation of OXPHOS, leading to reductions of cytoplasmic citrate, Ac-CoA, and acetylated histone levels. We also show that reduction in Ac-CoA by Acly deletion leads to a growth plate and molecular phenotype similar to that of LDH deficiency without inducing energy deficit, suggesting that the reduced level of Ac-CoA, but not ATP synthesis, is responsible for resting chondrocyte proliferation.

Mice
The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Massachusetts General Hospital.
Floxed Ldha mice were previously described 14 and purchased from the Jackson Laboratory. Floxed Acly mice were previously described 15 . Transgenic mice with a construct of Credependent expression of a constitutively active Fgfr3 mutant (Tg(CAG-Fgfr3*K650E,-EGFP)10Jheb) 16 were purchased from the Jackson Laboratory. Col2-Cre transgenic mice and Sox9-CreER transgenic mice were described 17; 18 . Ldhb knockout (Ldhb -/-) mice were generated by the CRISPR-mediated in vivo genome editing, i-GONAD 19 using the CD-1 outbred strain (Charles River Laboratory). Briefly, at 0.7 day post coitum, the oviduct of female mice was exposed through a dorsal incision. Approximately 1 ul of CRISPR solution containing 1 ug/ul of Cas9 (Sigma or IDT) premixed with 10 mM two-piece guide RNA (crRNA and tracrRNA, gRNA) (IDT), was injected into the oviduct either by puncturing the oviductal wall or through the infundibulum. Then, in vivo electroporation was performed using the square pulse generator, BTX-820 with the electrode tweezers, CUY652P2.5X4 (Nepa Gene, Japan) with the setting of 50V, 5msec each pulse, 8 pulses, with 0.5 cm electrode gap. Pups were genotyped for the desired modification, then mated with wildtype mice to establish a line. The Ldhb gene was deleted by cleaving the genome at two sites using two gRNAs mixed with Cas9 to remove a 15kb long genomic region containing exons encoding more than 90% of the Ldhb coding sequence including the entire active site. The spacer sequences of these crRNAs are Ldhb-L 5'-CGCAAUGAGCUUCUCCUUAA-3' and Ldhb-R 5'-ACGAUGAGGUCGCUCAGCUC-3'. Genotyping of the Ldhb gene was performed by PCR using the wildtype-specific primer set, Ldhb-L 5'-CCTTCAGGGCTTCTGTTGAG -3' and Ldhb-R 5'-CATGTCAGGGAAGAAGCAAA -3', and the KO-specific primer set, Ldhb-L and Ldhb-Wt 5'-CGCCCACTACAGTGATCTTG -3'. Mir140UGCG mice that express a gain-of-function mutation of the miR-140-5p gene (Mir140G, miR-140-5pG) 3 with additional mutations were described 20 . The additional mutations (the first nucleotide and last two nucleotides of miR-140-5p sequence of Mir140) facilitate the loading of miR-140-5p onto Argonaute proteins, and thereby enhances the expression of miR-140-5p-G and reduces miR-140-3p expression. These mutations do not alter the seed sequence and therefore do not significantly change its target RNA repertoire. Mir140UGCG mice show a greater gain-of-function effect of miR-140 mutation than Mir140G at the heterozygous state.
We found that Col2-Cre:Ldha fl/fl mice in the C57/B6 background were mostly perinatally lethal but that they survived postnatally in a mixed background of CD1 and C57/B6. Therefore, the experiments in this study were performed in a CD1-dominant congenic background.

Histological analyses
Mice were sacrificed at indicated ages. Tissues were dissected and fixed in 10% Formaldehyde/PBS for histological analysis. Decalcification was performed in EDTA for 2 weeks on a necessary basis, and then tissues were processed in paraffin. Sections were cut, stained with Hematoxylin and Eosin (H & E), or subjected to other analyses according to the standard protocols.
EdU (5-ethynyl-2′-deoxyuridine) labeling assay was performed according to the instruction of EdU Assay / EdU Staining Proliferation Kit (ab219801, Abcam). Proliferating cells were labeled by injecting 10mg/kg BW EdU to mice intraperitoneally 2 hours before sacrifice. Tissues were fixed, paraffin-processed, and subjected to EdU staining and DAPI nuclear staining.

Primary cell Isolation and cell culture
Mice were sacrificed at P10. Primary rib chondrocytes were isolated from these mice as previously described with some modifications 21 . After overnight collagenase digestion, the cells were placed in 2 mL tubes, centrifuged, and the collagenase was removed from the cells. Cells were resuspended in DMEM, 10% FBS, and 1% P/S. Cells were passed through a 0.40 um strainer (Corning) into a 6-well plate (Corning). Cells were cultured in DMEM-containing 10% FBS and antibiotics, counted, and replated for subsequent assays. Primary growth plate chondrocytes were isolated as previously reported with modifications 21 . Cells were cultured in DMEM containing 4.5g/L of glucose and 10% fetal bovine serum (FBS) without lactate in a humidified 37ºC incubator, otherwise specified.

RNA-seq analysis
RNA-seq was performed using polyA-enriched RNA from primary rib chondrocytes isolated from P5 mice of Col2-Cre:Ldha fl/fl :Ldhb +/and wildtype littermate control or from E18.5 rib chondrocytes of Col2-Cre:Acly fl/fl embryos and Cre-negative littermate control. RNA-seq was performed by Beijing Genome Institute (BGI). RNA-seq data is available at Gene Expression Omnibus (GEO) with the accession number GSE192971.

Seahorse analysis
Net activities of glycolysis and mitochondrial respiration were assessed using the mitostress test using the Seahorse XFe96 analyzer (Agilent) according to the manufacturer's instruction. Briefly, primary chondrocytes or bone marrow stromal cells, prepared as described above were . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 18, 2023. ; https://doi.org/10.1101/2023.01.18.524550 doi: bioRxiv preprint plated at the near confluence and cultured overnight in a growth medium (DMEM containing 10% FBS) before subjecting them to analysis.

Lipid analysis
Metabolites were assayed as previously described 22; 23 . In brief, Lipids were extracted from primary chondrocytes in monolayer culture in isopropanol and quantified using C8 chromatography and nontargeted, positive ion mode MS analysis on an Exactive Plus Orbitrap Mass Spectrometer. Identification of known metabolites was achieved by matching retention times and mass/charge ratio (m/z) to synthetic mixtures of reference compounds and characterized pooled plasma reference samples. Values were normalized by the cell mass. Lipid species were categorized into 6 groups. The average value of each lipid species was calculated, and the fold difference between control and mutant chondrocytes was plotted.

Statistical Analysis
Statistical analysis was performed using Prism 9 (GraphPad) . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 18, 2023. ; https://doi.org/10.1101/2023.01.18.524550 doi: bioRxiv preprint

Enhanced Expression of a Gain-of-Function Mutation of Mir140 Causes expansion of the Resting Zone of the Growth Plate.
The Mir140 gene encodes three microRNAs (miRNAs), miR-140-5p, miR-140-3p.1 and miR-140-3p.2 that are generated from a single pre-miR-140 hairpin precursor upon cleavage by the RNase III, Dicer. Then either the -5p or a -3p miRNA is selected and loaded onto Argonaute (Ago) proteins 24 . In chondrocytes, the abundance of miR-140-3p miRNAs, miR-140-3p.1 and -3p.2, is about 10 times greater than that of miR-140-5p likely due to asymmetric selection of miR-140 duplex strands during Ago loading. This strand selection appears to generally follow simple rules; the strand that starts uridine (U) or adenosine (A) is preferred for loading that those with guanosine (G) or cytidine (C), and the strand of which 5' end is located at the more thermodynamically unstable side of a duplex is preferred 25; 26 .
We previously reported that a single nucleotide substitution of the MIR140 in humans caused ultra-rare skeletal dysplasia, spondyloepiphyseal dysplasia, Nishimura type (SEDN, also known as SED, MIR140 type; MIM, 611894) 3 . In this condition, the single nucleotide substitution at the second nucleotide of miR-140-5p miRNA, from adenosine (A) to guanosine (G), causes a gainof-function (GOF) effect, leading to skeletal dysplasia both in humans and mice. In the mouse model carrying the identical substitution as in patients, the resting zone of the growth plate was expanded, causing a delay in ossification and development of the epiphysis.
The Mir140 GOF mutation showed a dose-dependent effect; homozygotes show a more severe phenotype than heterozygotes. To create a more robust mouse model, we introduced minor changes in the nucleotides at the 5' end of miR-140-5p and -3p to increase the expression level of miR-140-5p; the substitutions from C to U at the 5'-end of miR-140-5p and the change from UA to CG at the 5'-end of miR-140-3p increase the Ago loading efficiency of the -5p strand, thus enhances the GOF effect 20 ( Figure 1A). As expected, the mutant mice expressing the GOFmutant miR-140-5p (miR-140-5pG) with enhanced expression (Mir140UGCG) showed a significant expansion of the growth plate even in the heterozygous state, particularly in the resting zone in the tibia ( Figure 1B) and the bi-directional growth plate in the basal skull, the spheno-occipital synchondrosis ( Figure 1C). EdU labeling revealed a significant increase in cell proliferation in resting zone chondrocytes but not in proliferating chondrocytes ( Figure 1D).
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Decreased Glycolysis and Increased Oxidative Phosphorylation in Mir140UGCG Chondrocytes
To understand the mechanism by which the GOF mutant miR-140-5p (miR-140G) stimulates resting zone chondrocyte proliferation, we performed RNA-seq using primary rib chondrocytes of P7 mice of the previous mouse model without 5' nucleotide changes 3 . We found that the expression of genes whose products of which function is related to energy metabolism was significantly altered; those that regulate glycolysis was reduced, whereas those that regulate mitochondrial function and metabolism were reciprocally upregulated in a dose-dependent manner (Figure 2A, B). The upregulation of genes whose products are associated with glutaminolysis and lipolysis also suggested an increase in TCA cycle metabolism. Using primary chondrocytes, we performed the Seahorse analysis to assess the glycolysis and mitochondrial activity. Oxidative phosphorylation (OXPHOS) was assessed by real-time measurement of oxygen consumption rate (OCR) and glycolysis was assessed by extracellular acidification rate, a surrogate index for lactate production. The results supported the notion of reciprocal changes in glycolysis and mitochondrial metabolism, suggested by the RNA-seq data. ( Figure 2C). In our previous study, we identified a few direct targets of miR-140G including Hif1a. Hif1a is a major regulator of cellular energy metabolism in hypoxic conditions by stimulating glycolysis and suppressing mitochondrial metabolism 6; 12 . Thus, suppression of de novo targets of miR-140G directly and indirectly reduces glycolysis, a major mechanism for energy production in chondrocytes 10 , and increase mitochondrial metabolism to compensate for the energy deficit.
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Genetic Ablation of LDH Genes Increases Resting Chondrocyte Proliferation
Alterations of energy metabolism can have significant impacts on the regulation of cellular functions. Particularly, TCA cycle metabolites, such as acetyl CoA (Ac-CoA) and aketoglutarate (aKG), are known to play diverse regulatory roles. Thus, we hypothesized that altered energy metabolism contributed to the growth plate phenotype of Mir140G mutant mice. To test this hypothesis, we suppressed glycolysis by genetically ablating lactate dehydrogenase (LDH) genes. By catalyzing the conversion from pyruvate and lactate, LDH oxidizes nicotinamide adenine dinucleotide (NAD) + hydrogen (NADH) to generate NAD + , which is a cosubstrate of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), one of the key enzymes of glycolysis. Therefore, reducing LDH action is expected to suppress glycolysis. To do so, we ablated Ldha, the most abundantly expressed LDH gene, in chondrocytes using Col2-Cre transgenic mice. A majority of Col2-Cre:Ldha fl/fl (Ldha cKO) mice in the C57BL/6 background died at birth. However, in a congenic strain derived from the C57BL/6 and CD-1 backgrounds, Ldha cKO mice survive postnatally and grew into adulthood. Ldha cKO mice develop deformed and short long bones ( Figure 3A). Histological analysis revealed bowing of the tibia, increased cellular density of the resting zone, and loss of flattening of proliferating columnar chondrocytes ( Figure 3B). The proliferation of resting chondrocytes, but not proliferating columnar chondrocytes, was significantly increased in the Ldha cKO tibia ( Figure 3C, D). In the sphenooccipital synchondrosis, a bidirectional growth plate in the basal skull, showed an expansion of the resting zone ( Figure 3E). The expansion of the resting zone is also shared by Mir140G mutant growth plate, suggesting that the change in cellular metabolism is an underlying mechanism for the increased proliferation and expansion of resting chondrocytes.
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Overlapping Role of Ldha and Ldhb
There are 4 genes that encode LDH in mice. According to our RNA-seq data 3 , Ldhb is the second most abundantly expressed gene in chondrocytes next to Ldha, whereas Ldhc and Ldhd are hardly expressed. According to the International Mouse Phenotyping Consortium (IMPC, www.mousephenotype.org) database, Ldhb-null mice do not show overt skeletal abnormalities. To test whether Ldhb has an overlapping role with Ldha in chondrocytes, we generated Ldhbnull mice and cross them with Ldha cKO mice. An Ldhb germline-null mouse line was generated by i-GONAD in the congenic background as Ldha cKO mice. Both heterozygotes and homozygotes for Ldhb-null mice appear normal and fertile. However, compound mutant mice missing Ldha in chondrocytes and one allele of Ldhb (Col2-Cre:Ldha fl/fl :Ldhb +/-, Ldh compound cKO) showed a significantly smaller body size and growth defect compared with Ldha single conditional null mice (Col2-Cre:Ldha fl/fl ), and they usually died by 3 weeks of age ( Figure 4A, B). We never recovered live doubly homozygous null mice (Col2-Cre:Ldha fl/fl :Ldhb -/-), suggesting that complete loss of Ldha and Ldhb in chondrocytes results in embryonic or perinatal lethality. The shortening and bowing of long bones are more pronounced in Ldh compound cKO mice than Ldha single cKO mice ( Figure 4C). Histologically, both Ldha cKO mice and compound mutant mice showed shortening of the growth plate, particularly the hypertrophic zone, and this phenotype is a little more severe in the compound mutants ( Figure  4D). Using the Ldh compound cKO mutant mice, we performed Seahorse analysis to assess the impact of reduced LDH function in chondrocytes on energy metabolism. Ldh cKO chondrocytes showed mild downregulation of extracellular acidification rate (ECAR), a surrogate for lactate production, and reciprocal upregulation of oxygen consumption rate (OCR), an index of mitochondrial respiration. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 18, 2023.

Reduction in Acetylation in Mir140UGCG and Ldh cKO Mice
The reduced glycolysis, a predominant mechanism that synthesizes ATPs in chondrocytes, likely led to compensatory upregulation of mitochondrial OXPHOS in Mir140UGCG, which then would increase the retention of TCA cycle metabolites in mitochondria and therefore reduces their export into the cytoplasm. Consistent with this notion, the cytoplasmic concentration of Acetyl-CoA (Ac-CoA), generated mainly from citrate derived from mitochondria, as well as the cytoplasmic citrate concentration, was significantly reduced in Mir140UGCG chondrocytes ( Figure 5A). Because the availability of Ac-CoA in the cytoplasm and nucleus greatly influences acetylation of proteins, we evaluated the acetylation of histone proteins, the most abundant acetylated proteins in the cell ( Figure 5B, C). As expected, the levels of acetylated histones in Mir140UGCG primary chondrocytes were reduced in mutant chondrocytes. Since a similar bioenergetic alteration is induced by Ldha ablation, we assessed histone acetylation in Ldha cKO chondrocytes. We also found reductions in histone acetylation in Ldha-deficient cells ( Figure 5D, E).
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Skeletal Defects in Acly-Deficiency
Since the suppressed glycolysis is linked to reduced histone acetylation in Mir140UGCG and Ldha cKO mice, we hypothesized that reduced Acetyl-CoA (Ac-CoA) availability could be responsible for part of the skeletal phenotype in these mice. Cytoplasmic and/or nuclear Ac-CoA is generated from multiple sources, including citrate via the ATP-dependent citrate lyase, Acly, from dietary acetate via Acetyl-CoA synthases, and possibly from pyruvate via cytoplasmic/nuclear Pyruvate dehydrogenase (PDH), (PMID: 26039447). To mimic the citrate deficiency in Mir140UCGC mice, we conditionally ablated Acly in chondrocytes to reduce Ac-CoA availability. Conditional Acly-deficient (Col2-Cre:Acl fl/fl , Acly cKO) mice die at birth. The mutant long bones are short and bowed, which is reminiscent of the bone phenotype of Ldha or Ldha:Ldhb cKO mice ( Figure 6A). Histological examination of the tibial growth plate also revealed the impairment of flattening of Acly cKO columnar proliferating as in Ldha cKO mice. In the spheno-occipital synchondrosis, Acly cKO mice show an expansion of resting chondrocytes, a growth plate phenotype shared by Ldha cKO and Mir140UGCG mice ( Figure  6B). As expected Acly-deficient primary rib chondrocytes showed reduced levels of acetylated histones ( Figure 6C). Interestingly, phosphorylation of AMPK was not altered in Acly cKO mice, unlike Ldh cKO chondrocytes, suggesting that ATP synthesis is not affected by Acly-deficiency ( Figure 6D).
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Gene Expression Analysis in Ldh cKO and Acly cKO Chondrocytes
The phenotypic similarity between Ldha cKO and Acly cKO mice, including the shortening and bowing of long bones, expansion of resting chondrocytes, and reduced flattening of proliferating chondrocytes, prompted us to compare these two models at the molecular level. We performed RNA-seq analysis using P10-old primary rib chondrocytes of Ldh cKO chondrocytes and E18.5 Acly cKO rib chondrocytes. Because of the perinatal lethality of Acly cKO mice, we chose the late embryonic stage for chondrocyte isolation. Gene expression changes were evaluated by comparing mutants and littermate controls in these models. The average fold change of each gene was calculated in two models and plotted ( Figure 7A). We found a mild direct relationship in the changes in gene expression between Ldh cKO and Acly cKO chondrocytes. Pathway analysis on the most significantly altered gene sets showed overlapping ontology terms between these two models, suggesting, Ldh and Acly gene perturbations cause similar molecular consequences ( Figure 7B). Among these genes that are most significantly changed in both models, we found upregulations of groups of genes encoding chondrocyte-associated matrix proteins and downregulation of genes associated with endoplasmic reticulum (ER) stress ( Figure 6C). Additionally, we found upregulation of Fgfr3 that was included in the group with the term, "PI3K/AKT signaling pathway". Interestingly, the downregulation of ER stress genes and upregulation of Fgfr3 were also found in Mir140G chondrocytes.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 18, 2023. ; (C) Differentially expressed genes associated with indicated terms. Genes encoding chondrocyte-specific matrix proteins are generally upregulated both in Ldh and Acly cKO chondrocytes, whereas those associated with the term "ER stress" are downregulated. Expression of Fgfr3, associated with the term, "PI3K/Akt signaling" is increased in Ldh and Acly cKO chondrocytes. Downregulation of "ER stress"-associated genes and upregulation of Fgfr3 is also found in Mir140G/G chondrocytes. The gene expression data of Ldh and Acly cKO, and Mir140G/G is found at the Gene Expression Omnibus with Accession # GSE192971 and # GSE98309, respectively.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Colored bars indicate, the total growth plate length (blue), the combined length of the resting and proliferating zones (red), and the resting zone (green).
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Effects on Lipid Metabolism
Ac-CoA deficiency can also negatively affect lipid synthesis. We extracted lipid species from primary Acly cKO and Mir140G chondrocytes and subjected them to mass spectrometry analysis ( Figure 9A, B, Supple Table). Each lipid species was quantified and categorized into 6 groups, and the fold differences between control and mutant chondrocytes were plotted. Most lipid groups except the cholesterol ester group were not much different. Cholesterol esters, mostly present as lipid storage in the cell, were more abundant in Acly cKO chondrocytes and were less abundant in Mir140UGCG chondrocytes. This may be due to the possible increase in beta-oxidation in Mir140UGCG chondrocytes to compensate for the impaired ATP synthesis. Ras prenylation can be one of the indicators for the de novo lipid synthesis pathway, as prenyl pyrophosphates are intermediate metabolites in the cholesterol synthesis pathway, and protein prenylation plays an important regulatory role in several signaling pathways 27 . Interestingly, Ras prenylation was unaffected in Acly or Ldh cKO chondrocytes while Lovastatin treatment that suppresses the cholesterol synthesis pathway reduced it in primary chondrocytes ( Figure  9C). This result suggests that alternative pathways, particularly the Acss2-mediated Ac-CoA synthesis from acetate, compensate for the reduced Ac-CoA synthesis from citrate in these mutant chondrocytes for lipid synthesis reasonably well but less so for histone acetylation. (C) Ras prenylation was assessed by immunoblot analysis using whole cell lysates. Prenylated Ras proteins migrate faster due to subsequent processing. Whereas Lovastatin treatment to suppress the cholesterol synthesis pathway decreased prenylation, it was unaffected in Ldh-or Acly-deficient chondrocytes.
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Discussion
In this study, we show that the disease-causing GOF mutant miR-140 decreases glycolysis and increases OXPHOS in growth plate chondrocytes, that reduced glycolysis via Ldh ablation increases resting chondrocyte proliferation and reduces histone acetylation, and that Acly disruption causes cellular and molecular abnormalities similar to those of Ldh ablation in chondrocytes. These findings suggest that suppression of glycolysis stimulates resting chondrocyte proliferation by reducing the cytoplasmic/nuclear Ac-CoA availability.
The mechanism by which the proliferation of resting chondrocytes is regulated is poorly understood. Mir140G/G and Mir140UGCG mouse models with the unique growth plate phenotype, i.e., expansion of the resting zone, provided an opportunity to investigate the regulatory mechanisms that control resting chondrocyte proliferation. It was shown that Indian hedgehog (Ihh) signaling increases the proliferation of resting chondrocytes 28 . However, it does not cause an expansion of the resting zone; therefore the increase in proliferation may be a secondary consequence of the stimulated differentiation of resting chondrocytes into columnar proliferating chondrocytes 29 . In non-chondrocytic skeletal progenitor cells, Ras signaling stimulates the proliferation of Col2-positive osteochondro-progenitors in the bone marrow 30 . Our data that activation of Fgfr3 signaling causes an expansion of the resting zone of the growth plate supports the idea that the Ras signaling pathway might be a common regulator of skeletal stem/progenitor cell proliferation.
The findings from Mir140G, Ldh cKO, and Acly c KO mice, suggest a regulatory role of energy metabolism in controlling resting chondrocyte proliferation and differentiation of columnar proliferating chondrocytes. Because Acly cKO chondrocytes do not show increases in phospho-AMPK unlike Mir140G or Ldh cKO chondrocytes, energy deficiency is an unlikely cause for the phenotype. On the other hand, reduced histone acetylation was commonly observed in these three models, suggesting that the decreased Ac-CoA availability is responsible for the phenotype. Ac-CoA serves as the donor of the acetyl group for protein modification and also as a precursor molecule for de novo lipid synthesis. Our lipid quantification and assessment of Ras prenylation to indirectly assess the cholesterol synthesis pathway in primary chondrocytes from these mice showed no overt deficits, suggesting that lipid synthesis is not severely affected, unlike histone acetylation.
The increased expression of Fgfr3 commonly observed in these models and the stimulatory effect of Fgfr3 signaling in resting zone chondrocytes are consistent with the hypothesis that upregulation of Fgfr3 can be the underlying cause for the expanded resting zone. However, the mechanism by which Ac-CoA deficiency can lead to the Fgfr3 upregulation and these phenotypic changes is not clear at the moment. The decrease in histone acetylation suggests that the reduced availability of Ac-CoA could epigenetically alter gene expression including Fgfr3 or functions of undetermined regulatory proteins. For example, Hif1a acetylation regulates its stability and transcriptional activity 31; 32 .
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 18, 2023. ; https://doi.org/10.1101/2023.01.18.524550 doi: bioRxiv preprint Together, these results suggest that chondrocyte bioenergetics regulate gene expression by changing Ac-CoA metabolism, which differentially controls resting and proliferating chondrocytes possibly through FGFR3 signaling ( Figure 10A, B). Currently, firm evidence for upregulation in FGFR3 signaling or epigenetic deregulation of the Fgfr3 gene in these mouse models is missing; the proposed mechanism remains hypothetical.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 18, 2023. ; https://doi.org/10.1101/2023.01.18.524550 doi: bioRxiv preprint