Poster Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

INTRODUCTION

Histone post-translational modifications play important roles in epigenetic regulation and biomarker profiling for aging and age-related diseases. Lactate was recently discovered as a novel substrate for post-translational modification on histone lysine residues, known as histone lactylation¹. The intervertebral disc is hypoxic and produces abundant lactate as an end-product of anaerobic respiration. Historically assumed to be a toxic metabolic waste, lactate has recently been demonstrated in our lab to have a role in disc histone lactylation. However, whether histone lactylation regulates gene expression and what principal histone lysine lactyltransferases (lactate ‘writers’) exist in the intervertebral disc are still unknown. In this study, we aim to determine in AF cell culture model under normal and inflammatory conditions if 1) histone lactylation regulates expression of collagen1 (Col1), the principal matrix protein in AF, and 2) if MOF, CBP, and p300 are candidate cognate lactate writers by RNA silencing approach.

METHODS

AF tissues were isolated from spines of 3-month-old F344 rats as approved by the University of Pittsburgh's IACUC and expanded. Before being treated with different experimental conditions, P1 AF cells were conditioned in DMEM containing 1mM Glucose and 1% FBS at 5% O2 to mimic disc physiological nutrient niche for 24 hours. For concentration-dependent lactylation, cells were treated for 24 hours at 0, 2, 4, 10, and 20 mM lactate before protein collection. For gene silencing, AF cells were treated with MOF, CBP, and p300 siRNA (Horizon Discovery, Cat. M-087981-01-0005, Cat. L-091740-02-0005, Cat. L-101658-02-0005) and lipofectamine 3000 (Invitrogen). Cells were then exposed to high lactate conditions and -/+ IL-1β (5ng/ml) to model IDD inflammatory conditions. Col1 gene expression was evaluated using RT-PCR. Histone lactylation (Kla) was quantified using western blot. Membranes were blotted to detect Pan Kla (Cat. PTM-1401, PTM Biolabs) and Pan Kac (histone acetylation, Cat. PTM-101, PTM Biolabs). Histone H3 (Cat. PTM-1001, PTM Biolabs) was used as a control.

RESULTS

Histone lactylation, but not acetylation, was substantially induced in AF cell culture grown at 10 and 20 mM lactate compared to 1 mM lactate (p<0.05) (Figure 1). Col1 gene expression was significantly increased (p < 0.05) at 10 mM lactate compared to 0 mM lactate under both inflammatory and noninflammatory conditions (Figure 2). MOF, CBP, and p300 were all silenced with at least 80% transduction efficiency (Figure 3). Col1 expression was not affected by MOF silencing. With CBP silencing, Col1 expression was significantly decreased (p<0.05) in both inflammatory and noninflammatory conditions (Figure 3). Col1 decreased with p300 silencing only under inflammatory conditions.

DISCUSSION

Histone lactylation is induced by lactate in a dose-dependent manner in rat AF cell culture. Regulation of Col1 gene expression by lactate and specific writers (CBP, p300) suggest that histone lactylation is an important epigenetic regulator of this key matrix gene in AF in both noninflammatory and inflammatory conditions. We are currently conducting western blots to confirm if Col1 gene expression decrease is due to decreased histone lactylation after CBP silencing. Future work includes ChIP sequencing to determine lactylation involvement in epigenetic regulation of ECM homeostasis.