Objective: To study the expression and function of sphingosine kinase (SphK) 1 and SphK2 in human keloid fibroblasts.
Methods: Specimens of keloid and surrounding normal skin were collected from 12 patients with keloid during operation. Primary fibroblasts were isolated, cultured, and randomly divided into 3 groups: normal skin group, keloid group, and keloid with transforming growth factor (TGF)-beta1 group cultured with TGF-beta1 for 48 h. Immunofluorescence technique was used to detect the location of SphK1 and SphK2 protein. Real-time PCR and Western blotting were used to measure the mRNA and protein expression levels of SphK1 and SphK2.
Results: Sphk1 protein was localized primarily in the nuclei of the fibroblasts, and Sphk2 protein was detected both in the cytoplasm and nuclei in the 3 groups. The mRNA and protein levels of Sphk1 in the keloid group were (0.0608 +/- 0.0190) and (0.8308 +/- 0.1093) respectively, both significantly higher than those of the normal skin group [(0.0383 +/- 0.0147) and (0.6800 +/- 0.1126) respectively, both P < 0.05], but significantly lower than those of the keloid fibroblasts with TGF-beta1 group [(0.0790 +/- 0.0280), P < 0.05, and (1.4267 +/- 0.1938), P < 0.01]. There was no significant differences in the Sphk2 mRNA and protein levels among these 3 groups (all P > 0.05).
Conclusions: Sphk1 plays a leading role in keloid pathogenesis. The SphK1 mRNA and protein levels are increased by TGF-beta1 stimulation in keloid fibroblasts, perhaps indicating that Sphk1 is involved in TGF-beta signal transduction pathway.
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