Unfolded protein response regulation in keloid cells.
BACKGROUND
Keloids are a common form of pathologic wound healing characterized by excessive production of extracellular matrix. The unfolded protein response (UPR) is a cellular response to hypoxia, a component of the wound microenvironment, capable of protecting cells from the effects of over-accumulation of misfolded proteins. Since keloids have hypersecretion of extracellular matrix, we hypothesized that keloid fibroblasts (KFs) may have enhanced activation of the UPR compared with normal fibroblasts (NFs).


METHODS
KFs and NFs were placed in a hypoxia chamber for 0, 24, and 48h. We also used tunicamycin to specifically up-regulate the UPR. UPR activation was assayed by PCR for xbp-1 splicing and by immunoblotting with specific antibodies for the three UPR transducers. Nuclear localization of XBP-1 protein in KFs was confirmed by immunofluorescence.


RESULTS
There is increased activation of XBP-1 protein in KFs compared with NFs following exposure to hypoxia. Pancreatic ER kinase (PERK) and ATF-6, two other pathways activated by the UPR, show comparable activation between KFs and NFs. We confirmed that there is enhanced activation of XBP-1 by demonstrating increased nuclear localization of XBP-1 using immunofluorescence.


CONCLUSIONS
In contrast to our initial hypothesis that keloids would have broad activation of the UPR, we demonstrate here that there is a specific up-regulation of one facet of the UPR response. This may represent a specific molecular defect in KFs compared with NFs, and also suggests modulation of the UPR can be used in wound healing therapy.
Butler
Paris D
PD
Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148, USA.
Wang
Zhen
Z
Ly
Daphne P
DP
Longaker
Michael T
MT
Koong
Albert C
AC
Yang
George P
GP
D004268:DNA-Binding Proteins / Q000378:metabolism; D005109:Extracellular Matrix / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D006801:Humans; D000860:Hypoxia / Q000503:physiopathology; D007627:Keloid / Q000378:metabolism / Q000503:physiopathology*; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000378:metabolism; D056811:Unfolded Protein Response / Q000502:physiology*; D014945:Wound Healing / Q000502:physiology; D000071717:X-Box Binding Protein 1
D004268:DNA-Binding Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human