Differential expression and regulation of TGF-beta1, TGF-beta2, TGF-beta3, TGF-betaRI, TGF-betaRII and TGF-betaRIII in cultured human corneal, limbal, and conjunctival fibroblasts.
OBJECTIVE
We have reported that three patterns of cytokine expression are potentially involved between epithelia and fibroblasts of the human ocular surface. The TGF-beta family is a prototypical fibrogenic cytokine responsible for fibroblast activation in wound healing. We investigated how the TGF-beta family is differentially expressed and regulated in cultured human corneal, limbal and conjunctival fibroblasts.


METHODS
Human corneal (HCF), limbal (HLF) and conjunctival fibroblast (HJF) were cultured in DMEM-10% FBS until confluence and switched to serum-free DMEM-ITS for 48 h before adding 10 ng/ml of each of eight cytokines for 4 h in three separate experiments. Total RNA was isolated and subjected to Northern hybridization with GAPDH as a control. ELISA was used to determine TGF-beta1 and TGF-beta2 proteins in the media.


RESULTS
All three isoforms of TGF-beta and three types of TGF-betaR were expressed by HCF, HLF and HJF. Expression of TGF-beta1 mRNA was strongest and upregulated by the three TGF-betas in all three types of fibroblast. PDGF-BB and TGF-alpha slightly increased TGF-beta1 mRNA. TGF-betas also upregulated TGF-beta3 mRNA in HJF. TGF-betaRI mRNA was the only receptor upregulated by TGF-betas. TGF-betaRII and TGF-betaRIII mRNA were not regulated by all cytokines tested.


CONCLUSIONS
TGF-betas auto-induction is the major mechanism upregulating TGF-beta1 expression. Promotion of TGF-beta3 by the TGF-betas may have a special role in HJF. Differential expression and regulation of TGF-betas and TGF-betaRs suggest that each TGF-beta isoform may have specific functions in different ocular surface fibroblasts. No cytokine tested can downregulate TGF-beta1 and the TGF-betaRs.
Ocular Surface and Tear Center Department of Ophthalmology Bascom Palmer Eye Institute USA Florida, Miami, FL 33101, USA.
D Q
DQ
Li
S B
SB
Lee
S C
SC
Tseng
D000328:Adult; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000378:metabolism*; D003315:Cornea / Q000166:cytology / Q000378:metabolism*; D016207:Cytokines / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D012590:Sclera / Q000166:cytology / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; D020033:Protein Isoforms; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta