Insulin-like growth factor-I is expressed by avian flexor tendon cells.
Cells in normal tendon are in a resting G0 state, performing maintenance functions. However, traumatic injury introduces growth factors such as platelet-derived growth factor and insulin-like growth factor from blood as well as activates endogenous growth factors. These factors stimulate migration and proliferation of tendon cells at the wound area. Tendon cells require growth-promoting factors to transit the cell cycle. To evaluate the contribution of endogenous growth factors in tendon, extracts of the epitenon and internal compartment of avian flexor tendon as well as medium of cultured cells from the epitenon (tendon surface cells) and internal tendon (tendon internal fibroblasts) were collected to assess their ability to stimulate DNA synthesis. Acid-ethanol extracts of tissues and medium were chromatographed on a P-30 molecular sieve column and assayed for mitogenic activity by quantitating [3H]thymidine incorporation into tendon cell DNA. The extract from the internal tendon compartment was more stimulatory for DNA synthesis than that from the epitenon, particularly when tested on tendon internal fibroblasts. However, conditioned medium fractions from surface epitenon cells stimulated DNA synthesis to a high degree on both tendon surface cells and tendon internal fibroblasts. Conditioned medium from tendon internal fibroblasts was also stimulatory. An anti-insulin-like growth factor-I antibody ablated most of the mitogenic activity present in both tissues and conditioned medium. The levels of acid-extractable insulin-like growth factor-I in tendon were determined by competitive radioimmunoassay as 1.48+/-0.05 ng/g tissue for the epitenon and 3.83+/-0.03 ng/g tissue for the internal compartment. Results of Western immunoblots of conditioned medium revealed insulin-like growth factor-I at the 7.5 kDa position. Cultured tendon surface cells and tendon internal fibroblasts as well as cells in intact flexor tendon expressed insulin-like growth factor-I mRNA detected by reverse transcriptase-polymerase chain reaction. In situ hybridization histochemistry positively identified insulin-like growth factor-I mRNA in tendons from 52-day-old chickens. Platelet-derived growth factor was not detected at the protein or message levels. Furthermore, tendon surface cells and tendon internal fibroblasts both expressed receptors for insulin-like growth factor-I detected by flow cytometry. These data suggest that tendon cells express insulin-like growth factor-I mRNA and synthesize insulin-like growth factor-I in both the epitenon and the internal compartment of tendon, which is present in an inactive form, most likely bound to insulin-like growth factor-binding proteins.
Tsuzaki
M
M
Department of Orthopaedics, University of North Carolina School of Medicine, Chapel Hill 27599-7055, USA.
Brigman
B E
BE
Yamamoto
J
J
Lawrence
W T
WT
Simmons
J G
JG
Mohapatra
N K
NK
Lund
P K
PK
Van Wyk
J
J
Hannafin
J A
JA
Bhargava
M M
MM
Banes
A J
AJ
D000818:Animals; D000906:Antibodies; D000077214:Becaplermin; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002457:Cell Extracts / Q000494:pharmacology; D002478:Cells, Cultured; D002645:Chickens; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005434:Flow Cytometry; D015870:Gene Expression / Q000502:physiology; D007334:Insulin-Like Growth Factor I / Q000032:analysis* / Q000235:genetics* / Q000276:immunology; D010982:Platelet-Derived Growth Factor / Q000032:analysis / Q000276:immunology; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger / Q000032:analysis; D013708:Tendon Injuries / Q000503:physiopathology; D013710:Tendons / Q000737:chemistry* / Q000166:cytology / Q000502:physiology*; D014945:Wound Healing / Q000502:physiology
D000906:Antibodies; D002457:Cell Extracts; D017077:Culture Media, Conditioned; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D000077214:Becaplermin; D007334:Insulin-Like Growth Factor I