Reduced PPOX Expression Causes Intrinsic Pathogenicity in Keratinocytes, Contributing to the Cutaneous Variegate Porphyria Phenotype

Variegate porphyria, caused by monoallelic variants in PPOX (protoporphyrinogen oxidase), leads to acute visceral attacks and skin photosensitivity, presenting as blistering and fragility; a more severe phenotype occurs with rare biallelic variants. This results from accumulation of intermediate porphyrins, such as 5'-aminolevulenic acid, near the skin surface, oxidized by sunlight and releasing free radicals. We hypothesized that reduced PPOX expression in keratinocytes intrinsically contributes to the porphyria phenotype. We created 2 short hairpin RNA-knockdown cell lines, KD1 and KD2 (50 and 25% residual expression), to simulate haploinsufficiency and biallelic hypomorphic expression. Both knockdown lines showed significantly reduced proliferation, with KD2 also having reduced migration. Keratinocytes were treated with 5'-aminolevulenic acid and deferoxamine (an iron chelator) to accumulate porphyrins, confirmed by increased fluorescent downstream porphyrins, which caused reduced proliferation, oxidative stress, and a lower reduced glutathione/oxidized glutathione ratio. Both clones expressed reduced differentiation in monolayer and 3-dimensional cultures, with KD2 showing reduced epidermal thickness, whereas porphyrin accumulation further disrupted stratification and differentiation. We propose an updated paradigm of porphyria skin manifestations: porphyrin excitation beneath the skin is facilitated by a thinner, less differentiated barrier. These 3-dimensional skin models offer a translational platform to investigate therapeutic strategies targeting porphyrin accumulation and barrier restoration in porphyria.