Expression of PKM2 in wound keratinocytes is coupled to angiogenesis during skin repair in vivo and in HaCaT keratinocytes in vitro
Authors:   , Sych Khrystyna 
Published: December 2024

Skin is the largest organ of human body with protective, sensory as well as thermoregulatory capabilities. Therefore, it is of an utmost importance to maintain its integrity and proper function. An injured skin must be rapidly restored by means of wound healing, a complex, multi-staged, acute inflammatory manner.
Similar to a neoplasm, it is defined, at least in part, by the virtue of rapidly proliferating cells. However, as opposed to the uncontrolled cancerogenesis, wound repair is a well-coordinated process.
Former works from our group and others highlighted the importance of an intact insulin signalling and glucose utilisation for operational healing. This work interrogates the metabolic drivers of cutaneous wound healing, following the knowledge obtained from the research on tumor biology. In the 1920s Otto Warburg reported cancer cells to differ from non-transformed cells, by utilizing glycolysis instead of more efficient mitochondrial oxidative phosphorylation (OxPhos) for energy production, even in the presence of oxygen. This aerobic glycolysis, termed as the Warburg phenomenon, is taken over in rapidly proliferating cells e.g. activated lymphocytes. Indeed, genes related to central pathways of glycolysis, pentose phosphate shunt, OxPhos and replenishing of carbon in the mitochondrial citric acid cycle via glutamine anaplerosis, were found to be highly induced during the acute phase of wound inflammation. We were attracted by the induced expression of pyruvate kinase M2 (Pkm2) as this protein has been suggested to compel aerobic glycolysis in tumor cells.
Pyruvate kinase (PK) is the rate-limiting glycolytic enzyme, that catalyses the very last step of glycolysis. In vertebrates, two genes code for PK. PKLR encodes the PKL and PKR isoforms, expressed in the liver and reticulocytes, respectively.
PKM1 and PKM2 are variably expressed in the rest of the body’s tissues by the alternatively spliced mRNA variants of PKM gene, harbouring exon 9 or 10 respectively. Both exons, 9 and 10, are equally 167 nucleotides long and differ only in the coding sequences for 22 amino acid codons that determines the specificity of the two isoforms. While PKM1 is expressed as a constantly active homo tetramer, PKM2 appeared as metabolically inactive mono- or dimer and can be allosterically induced to form a metabolically active tetramer. The non-tetrameric forms of PKM2 have been implied in many “moonlight” metabolic and non-metabolic activities that foster neoplasia.
Pkm2 mRNA and protein is notably induced from day 3 with a peak on day 5 of injury and its expression is continuously high until wound closure, while the expression of Pkm1 remained unaltered. Furthermore, Pkm2 is present almost exclusively as a dimer, indicating a non-metabolic function during wound repair.
Immunohistochemistry staining showed that Pkm2 expression is mainly limited to immune cells (macrophages and neutrophils) and wound marginal keratinocytes.
Analysis of Vegf expression in the wound edge has shown its confinement to Pkm2- expressing keratinocytes facing the wound bed, implying a role of Pkm2 in Vegf expression in keratinocytes. Along this line, the expression of PKM2 has been previously shown to be upregulated by Hif-1α, which in turn enhances the expression of Hif-1α target genes (e. g. Vegf). Although Hif-1α staining has also been localized to the hyperproliferative epithelia (HE), it is sparsely expressed in keratinocytes lying adjacent to the wound edge, suggesting Vegf expression in wound keratinocytes is Hif1α-independent. Knockdown of PKM2 in HaCaT keratinocytes resulted in a substantial decrease of growth factor induced VEGF mRNA and protein expression, indicating PKM2 as an essential factor to induce angiogenesis by keratinocytes. Unexpectedly, knockdown of PKM2 in keratinocytes initially induces the protein translational machinery, perhaps as stress response to acute silencing of PKM2. However, the mid- to late-time point marked attenuation of S6 ribosomal protein (S6RP) activation, provides a mechanistic explanation to the decline in VEGF expression upon PKM2-silencing. Interestingly, knockdown of PKM2 increases cytokines- and growth factor activation of STAT3, as also manifested by induction of its suppressor, SOCS3. Since VEGF production as well as the induction of SOCS3 in EGF-treated keratinocytes is increased upon co-stimulation with IL-6, we assume no involvement of SOCS3 in the decreased expression of VEGF in PKM2-silenced cells. Pharmacological induction of PKM2 tetramerization by small molecule activator TEPP-46, did not alter the induction of VEGF expression, suggesting a non-metabolic function of PKM2 to support angiogenesis by keratinocytes.
In conclusion, this work provides evidence that PKM2 is involved in the induction of angiogenesis by keratinocytes, facilitating maximum expression of VEGF upon inflammatory stimuli.