Niche-Driven Overgrowth of KPC Figure 5. IL-17-induced hTERT and Oct-4 expression is IL-6-dependent. A, IL-17 stimulated IL-6 secretion in SKPs and KPCs as determined by ELISA analysis (mean6SEM). ** P,0.01; ns, no significance. B, IL-17 stimulated the expression of hTERT and Oct-4 in SKPs and KPCs as determined by Western blot analysis. C, Blocking IL-6, but not IL-17 attenuated the basal expression of hTERT and Oct-4 in SKPs and KPCs. Cells were treated with 5 mg/mL of neutralizing antibody for either IL-6 (IL-6 Ab), or IL-17 (IL-17Ab), or both for 24 hours and expression of hTERT and Oct-4 was determined by Western blot analysis. D, IL-17-induced upregulation of hTERT and Oct-4 expression in SKPs and KPCs was significantly attenuated by treatment with neutralizing antibodies for either IL-17, or IL-6, or both. After serum-starved for 24 hours, cells were pretreated with neutralizing antibodies, followed by incubation with 20 ng/mL of IL-17. The expression of hTERT and Oct-4 was determined by Western blot analysis. E, IL-6-induced upregulation of hTERT and Oct-4 expression in SKPs and KPCs was attenuated only by IL-6Ab, not by IL-17Ab. Following serum-starvation for 24 hours, cells were pretreated with neutralizing antibodies and incubated with 20 ng/mL of IL-6. Expression of hTERT and Oct-4 was determined by Western blot analysis. An isotype-matched normal mouse IgG (mIgG) was used as negative controls. The results are representative of at least five independent experiments using KPCs and the matched SKPs from different patient donors (n = 5). doi:10.1371/journal.pone.0007798.g005 presence of IL-6 in hydrogel carrier significantly promoted the in vivo growth of both KPCs and SKPs (Figure 6A). Histological analyses revealed significant increase in cellular components and type I collagen expression in all transplants using IL-6-retained hydrogel as compared to hydrogel only (Figures 6B and 6C). The collagen structure of IL-6 treated transplant was more fibrillar and less amorphous. Importantly, abundant keloid-like thick collagen fibrils were observed in KPC transplants treated with IL-6 as demonstrated by histological and immunohistological staining (Figures 6B and 6C), and electron microscopy (Figure 6D). The unique pattern of excessive intracellular and extracellular accumulation of collagen seen in KPC transplants resembled the abundant collagen matrix observed in keloid tissues (Figure S1B). Meanwhile, we demonstrated that transplants generated from KPCs and SKPs in the presence of IL-6 expressed higher levels of Oct-4 and hTERT, as well as an elevated hTERT enzyme activity, as compared to control (Figures 6E, 6F and 6G). The increased proliferation of dermal stem cells by IL-6 was further confirmed by immunohistochemical staining with antibody for human PCNA (proliferating cell nuclear antigen) (Figure 6H). These results provide further evidence that IL-6, as a downstream target of IL-17 and an essential component of the unique PLoS ONE | www.plosone.org 10 November 2009 | Volume 4 | Issue 11 | e7798