Abstract
Transforming growth factor-βs (TGF-βs) signal after binding to the TGF-β receptors TβRI and TβRII. Recently, however, betaglycan (BG) was identified as an important co-receptor, especially for TGF-β2. Both proteins are involved in several testicular functions. Thus, we analyzed the importance of BG for TGF-β1/2 signaling in Sertoli cells with ELISAs, qRT-PCR, siRNA silencing and BrdU assays. TGF-β1 as well as TGF-β2 reduced shedding of membrane-bound BG (mBG), thus reducing the amount of soluble BG (sBG), which is often an antagonist to TGF-β signaling. Treatment of Sertoli cells with GM6001, a matrix metalloproteinases (MMP) inhibitor, also counteracted BG shedding, thus suggesting MMPs to be mainly involved in shedding. Interestingly, TGF-β2 but not TGF-β1 enhanced secretion of tissue inhibitor of metalloproteinases 3 (TIMP3), a potent inhibitor of MMPs. Furthermore, recombinant TIMP3 attenuated BG shedding. Co-stimulation with TIMP3 and TGF-β1 reduced phosphorylation of Smad3, while a combination of TIMP3/TGF-β2 increased it. Silencing of BG as well as TIMP3 reduced TGF-β2-induced phosphorylation of Smad2 and Smad3 significantly, once more highlighting the importance of BG for TGF-β2 signaling. In contrast, this effect was not observed with TIMP3/TGF-β1. Silencing of BG and TIMP3 decreased significantly Sertoli cell proliferation. Taken together, BG shedding serves a major role in TGF-β2 signaling in Sertoli cells.
Highlights
The Transforming growth factor-βs (TGF-βs) superfamily is a large group of proteins consisting of various TGF-β isoforms, bone morphogenetic proteins (BMPs), growth and differentiation factors, activins, inhibins, and glial-derived neurotrophic factors
We found that TGF-β2, in particular, significantly increased mRNA expression of BG after 24 h as well as after 48 h (Figure 1A,B)
This could not account for the functional diversity such as the importance of TGF-β1 in inflammation [42], the impact of TGF-β2 in several developmental defects [43] and TGF-β3-dependent deficiencies in lung and cleft palate [44] observed in vivo by silencing of the TGF-βs
Summary
The TGF-β superfamily is a large group of proteins consisting of various TGF-β isoforms, bone morphogenetic proteins (BMPs), growth and differentiation factors, activins, inhibins, and glial-derived neurotrophic factors. It is well established that TGF-βs are crucial during testis development [1,2,3]. TGF-βs affect the proliferation and apoptosis of testicular germ and somatic cells [4,5]. TGF-β isoforms are highly expressed on mRNA level in Sertoli cells and their expression changes during development [6,7]. The quantities of active TGF-β proteins secreted by Sertoli cells into the medium are rather low [8,9]. Sertoli cell proliferation and testis cord formation are regulated by TGF-β signaling [10]. TGF-β3 disturbs the blood–testis barrier possibly by targeting occludin, zonula occludens-1 and claudin-11 in junctional complexes [11]
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