Substance P increases CCN2 dependent on TGF-beta yet Collagen Type I via TGF-beta1 dependent and independent pathways in tenocytes

Abstract

Transforming growth factor beta 1 (TGFbeta-1) and connective tissue growth factor (CCN2) are important mediators of tissue repair and fibrosis, with CCN2 functioning as a downstream mediator of TGFβ-1. Substance P (SP) is also linked to collagen production in tenocytes. A link between SP, TGFbeta-1 and CCN2 has yet to be established in tenocytes or fibrogenic processes. We sought to determine whether SP induces tenocyte proliferation, CCN2, or collagen production via TGFbeta-1 signaling or independently in rat primary tenocytes. Tenocytes were isolated from rat tendons, cultured and stimulated by SP and/or TGFbeta-1. Cultured cells expressed proteins characteristic of tenocytes (vimentin and tenomodulin) and underwent increased proliferation dose dependently after SP and TGFbeta-1 treatments, alone or combined (more than SP alone when combined). SP induced TGFbeta-1 expression in tenocytes in both dose- and time-dependent manners. SP and TGFbeta-1, alone or combined, stimulated CCN2 expression in tenocytes and their supernatants after both 24 and 48 h of stimulation; a response blocked with addition of a TGFbeta-1 receptor inhibitor. In contrast, SP potentiated collagen type I secretion by tenocytes, a response abrogated by the TGFbeta-1 receptor inhibitor after 48 h of stimulation, but not after the shorter 24 h of stimulation. Our findings suggest that both SP and TGFbeta-1 can stimulate tenocyte fibrogenic processes, albeit differently. TGFbeta-1 pathway signaling was involved in CCN2 production at all time points examined, while SP induced collagen type I production independently prior to the onset of signaling through the TGFbeta-1 pathway.

Blocking CTGF/CCN2 reduces established skeletal muscle fibrosis in a rat model of overuse injury

“…CCN2/CTGF has been shown to modulate many signaling pathways leading to extracellular matrix deposition 46 . It plays a key role in matrix over‐production and is markedly elevated in fibrotic organs and tissues from animal models and humans with various fibrotic diseases ranging from hepatic fibrosis, renal disease with fibrosis, mesothelioma, idiopathic pulmonary fibrosis, muscle dystrophy, skin fibrosis, overuse injury, and more 24,26‐29,47 . CCN2 is a downstream mediator of TGFβ1, 48,49 and increases in tissues under conditions of overload 50 .…”

Fascial tissue research in sports medicine: from molecules to tissue adaptation, injury and diagnostics: consensus statement

“…Recent studies show that substance P can stimulate TGFβ-1 production by tendon fibroblasts, and that both substance P and TGFβ-1 can induce fibrogenic processes independently of each other. 56 …”

The answer depends on the question: Optimal conditions for western blot characterization of muscle collagen type 1 depends on desired isoform

“…The intact disulfide bonded collagen type 1 has a molecular weight range of ~250 and higher under non-reduced conditions (no BME) [7]. The observed molecular weight ranges of procollagen a1(I) and a2(I) chains are ~140–200 kDa [1,3,5,7,8]. Cleavage of first the N-terminus propeptide and then the C-terminus propeptide yields mature collagen a1(I) and a2(I) (~100 to 140 kDa dependent on tissue type, preparation and assessment conditions) [3,5,7,9,10].…”

“…Collagen a1(I) and a2(I) from which the N- and C-terminals have been removed are secreted and function as the building block for collagen fibril formation [11]. Further processing produces cleaved collagen I products that range between ~50–80 kDa in muscles [6,8].…”