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Paulette M. Robinson, Meera Dave, Tyler S. Smith, Alfred S. Lewin, Gregory S. Schultz; Identification of a 21 kDa Connective Tissue Growth Factor Fragment From Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2011;52(14):411.
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Stromal scarring due to corneal trauma, infection, or refractive surgery is the result of a complex cascade of multiple growth factors, cytokines, chemokines, and proteases. Connective tissue growth factor (CTGF) is a 38kDa fibrogenic cytokine that is a downstream mediator of many of the fibrotic actions of TGF-β. Grotendorst reported that a 18 kDa N-terminal fragment of CTGF predominately stimulated synthesis of ECM components and differentiation of cultured rat kidney fibroblasts into myofibroblasts, whereas the a C-terminal 20 kDa fragment preferentially stimulated proliferation. Low molecular weight bands have been detected in cell homogenates, biological fluids and tissue extracts by Western blots. We used tandem mass spectroscopy and western blots to positively identify a 21 kDa CTGF fragment in extracts of human corneal fibroblasts (HCF).
Cultures of HCF were serum starved for 48 hours then stimulated with 10 ng/mL of TGF-β1. After 24 hours, the cell extracts were removed and stored with a protease inhibitor cocktail. To determine if TGF-β1 stimulated HCF to process CTGF into fragments, extracts were concentrated and separated on a 12% SDS PAGE gel. Western blots were probed for CTGF with polyclonal antibodies. To further identify immunoreactive CTGF bands, HCF extracts were affinity purified using an anti-CTGF affinity column and eluted proteins were separated on a 12% SDS PAGE gel. Colloidal Coomassie stained bands that migrated and corresponded to the immunoreactive CTGF bands were excised and analyzed by LC/MS/MS.
Extracts of HCF stimulated by TGF-β1 contained two bands that were detected on Western blots at apparent molecular weights of 38 kDa and 21 kDa. The 21 kDa CTGF fragment and 38 kDa full length protein where both identified by the following sequence, LEDTFGPDPTMIR, that spans amino acids 184-196 of CTGF.
These results clearly show that full length (38 kDa) and a proteolytically processed fragment (21 kDa) of CTGF are present in the HCF cell extracts. The identification and purification of this fragment may allow for determination of the cleavage site of the full length CTGF. Proteolytic processing may be the mechanism that CTGF uses to elicit two completely independent cellular responses.
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