PKM2 mediates the glycolytic response to matrix stiffness in lung fibroblasts

Matrix stiffening during idiopathic pulmonary fibrosis (IPF) creates a mechanically altered microenvironment that promotes fibroblast activation, yet the metabolic consequences of these mechanical cues remain incompletely defined. Pyruvate kinase M2 (PKM2) catalyzes the rate-limiting step of glycolysis, but whether it serves as a mechanosensitive link coupling matrix stiffness to fibroblast activation has not been clarified. This study examined whether substrate stiffness drives glycolytic reprogramming through PKM2 upregulation and whether this metabolic adaptation is required for mechanically driven fibroblast activation.

PKM2 expression was analyzed in the NCBI GEO dataset GSE24206, human IPF lung tissues, and the bleomycin-induced mouse pulmonary fibrosis model. Primary mouse lung fibroblasts were cultured on collagen I-coated polyacrylamide hydrogels mimicking normal (2 kPa) or fibrotic (25 kPa) stiffness with or without TGF-β1 treatment. Glucose uptake, lactate secretion, pyruvate kinase activity, LDH activity, NAD⁺/NADH ratio, and oxygen consumption were measured; PKM2 oligomeric states were resolved by DSS cross-linking; and PKM2 was depleted by lentiviral shRNA.

PKM2 transcripts were progressively elevated in early- and advanced-stage IPF, and corresponding increases in protein and mRNA were confirmed in human IPF lungs and in bleomycin-treated mouse lungs. Stiff substrates upregulated PKM2 expression and reprogrammed fibroblast metabolism, with TGF-β1 eliciting increases in glucose uptake, lactate secretion, and oxygen consumption that were predominantly observed on stiff substrates. Cross-linking analysis showed that soft substrates favored catalytically active PKM2 tetramers, whereas stiff substrates constrained tetramer assembly and promoted lower-order species, providing a mechanistic basis for the dissociation between increased PKM2 expression and unchanged pyruvate kinase activity. PKM2 knockdown attenuated stiffness-induced increases in glucose uptake, lactate secretion, and LDH activity, reduced the cellular NAD⁺/NADH ratio, and decreased α-SMA-positive stress fiber formation under combined mechanical and TGF-β1 stimulation.

These findings indicate that PKM2 contributes to a mechanosensitive metabolic program coupling matrix stiffness to fibroblast activation, with substrate stiffness regulating both PKM2 expression and oligomeric state. Targeting PKM2-mediated metabolic adaptation may interrupt the self-reinforcing cycle of matrix stiffening and fibroblast activation in pulmonary fibrosis.