Targeting TGF-ß Mediated SMAD Signaling for the Prevention of Fibrosis.

Fibrosis occurs when there is an imbalance in extracellular matrix (ECM) deposition and degradation. Excessive ECM deposition results in scarring and thickening of the affected tissue, and interferes with tissue and organ homeostasis - mimicking an exaggerated "wound healing" response. Many transforming growth factor-ß (TGF-ß) ligands are potent drivers of ECM deposition, and additionally, have a natural affinity for the ECM, creating a concentrated pool of pro-fibrotic factors at the site of injury. Consequently, TGF-ß ligands are upregulated in many human fibrotic conditions and, as such, are attractive targets for fibrosis therapy. Here, we will discuss the contribution of TGF-ß proteins in the pathogenesis of fibrosis, and promising anti-fibrotic approaches that target TGF-ß ligands.

BMP15 Mutations Associated With Primary Ovarian Insufficiency Reduce Expression, Activity, or Synergy With GDF9.

CONTEXT: Bone morphogenetic protein (BMP)15 is an oocyte-specific growth factor, which, together with growth differentiation factor (GDF) 9, regulates folliculogenesis and ovulation rate. Multiple mutations in BMP15 have been identified in women with primary ovarian insufficiency (POI), supporting a pathogenic role; however, the underlying biological mechanism of many of these mutants remains unresolved. OBJECTIVES: To determine how mutations associated with ovarian dysfunction alter the biological activity of human BMP15. DESIGN: The effects of 10 mutations in BMP15 on protein production, activation of granulosa cells, and synergy with GDF9 were assessed. RESULTS: Sequencing of 35 patients with POI identified both an unrecognized BMP15 variant (c.986G>A, R329H) and a variant (c.581T>C, F194S) previously associated with the condition. Assessing expression and activity of these and 8 other BMP15 mutants identified: (1) multiple variants, including L148P, F194S, and Y235C, with reduced mature protein production; (2) three variants (R138H, A180T, and R329H) with ∼fourfold lower activity than wild-type BMP15; and (3) 3 variants (R68W, F194S, and N196K) with a significantly reduced ability to synergize with GDF9. CONCLUSIONS: Mutations in BMP15 associated with POI reduce mature protein production, activity, or synergy with GDF9. The latter effect is perhaps most interesting given that interactions with GDF9 most likely underlie the physiology of BMP15 in the human ovary.

A Novel, More Efficient Approach to Generate Bioactive Inhibins.

Gonadal-derived inhibins are essential factors in mammalian reproduction, negatively regulating pituitary production of FSH. Interestingly, declines in inhibin levels across the menopause transition correlate with not only an increase in FSH but also a rapid decrease in bone mass. Therefore, inhibins have been touted as potential therapeutics for osteoporosis in postmenopausal women. However, as heterodimeric proteins of α- and ß- (ßA or ßB)-subunits, inhibins are difficult to produce recombinantly, are poorly processed to their mature bioactive forms, and their expression is always accompanied by production of activins (ß-subunit homodimers), the proteins they antagonize. In this study, we developed the methodology to circumvent most of these issues. Initially, the cleavage sites between the pro- and mature domains of the α- and ßA-subunits were modified to ensure complete processing. These modifications led to a marked increase (9-fold) in the levels of bioactive inhibin A and a striking decrease (12.5-fold) in mature activin A production. Next, a single point mutation (M418A) was incorporated into the ßA-subunit, which reduced residual activin activity approximately 100-fold and, in so doing, increased inhibin bioactivity 8-fold. Finally, we showed that inhibin A noncovalently associated with its prodomain was more potent (∼20-fold) than mature inhibin A in specific in vitro bioassays, indicating an important role of the prodomain in inhibin bioactivity. In conclusion, the production of potent inhibin analogs in the virtual absence of activin activity will greatly facilitate the investigation of the therapeutic potential of these gonadal hormones on bone and other tissues.

Biological activity and in vivo half-life of pro-activin A in male rats.

Mature TGF-ß proteins are used in vivo to promote bone growth, combat obesity, reverse fibrosis and pulmonary arterial hypertension, and as potential rejuvenation factors. However, the serum half-life of this family of growth factors is short (∼5 min), limiting their therapeutic potential. Because TGF-ß proteins are normally secreted from cells with their prodomains attached, we considered whether these molecules could extend the in vivo half-life and activity of their respective growth factors. Using activin A as a model ligand, we initially modified the cleavage site between the pro- and mature domains to ensure complete processing of the activin A precursor. Co-immunoprecipitation studies confirmed mature activin A is secreted from cells in a non-covalent complex with its prodomain, however, the affinity of this interaction is not sufficient to suppress activin A in vitro biological activity. The plasma clearance profiles of purified pro- and mature activin A were determined over a 4 h period in adult male rats. Both activin forms demonstrated a two-phase decay, with the half-life of pro-activin A (t1/2 fast = 12.5 min, slow = 31.0 min) being greater than that of mature activin A (t1/2 fast = 5.5 min, slow = 20.3 min). Both pro- and mature activin A induced significant increases in serum follicle stimulating hormone levels after 4 h, but no differences were observed in the relative in vivo bioactivities of the two activin isoforms. Increased serum half-life of activin A in the presence of its prodomain identifies a new means to increase the therapeutic effectiveness of TGF-ß proteins.