The data presented in this work establish the anti-regenerative properties and inhibitory range of TGF-β1. These data also demonstrate that the age-related increase in TGF-β1 levels is conserved between mouse and human sera ( and ), and may therefore be universal to the aging process of mammals. In our studies, there was less variation in measurable TGF-β1 levels within mice (when compared with humans), likely owing to their environmental and genetic homogeneity. It is also important to note that our reported human TGF-β1 levels are representative of the healthy aging population, as individuals for these studies were prescreened for confounding medical conditions. Despite this, none of the aged individuals (≥ 60 years) had less than measurable levels of TGF-β1, as opposed to many of the young individuals (≤ 30 years) enrolled in these studies. Considering that TGF-β1 is broadly produced, and signals to a variety of cells, the age-related and evolutionarily conserved elevation of its functional levels in old sera may affect stem cell responses in various other tissues. Such a finding may provide a preliminary explanation for the general organ stem cell regenerative potential decline of mice and humans.
With respect to satellite cell activation, TGF-β1 operates in a threshold fashion (), and appears to function through the TGF-β II receptor and P-Smad signaling ( and ). Our findings suggest that certain levels of TGF-β1 are actually required for normal satellite cell responses (). Experimental down-modulation of this pathway is therefore expected to be typically harmful, unless it is transient (, ––) or performed to precise ‘beneficial’ levels. These conclusions fit well with studies, reporting that among other important functions, TGF-β1 signaling is needed for efficient immune cell and wound clearance responses (Dunker & Krieglstein, 2000
). TGF-β proteins have been shown to behave as morphogens which, depending on concentration, induce various gene subsets and promote different effects (McPherron et al., 1997
; Itoh et al., 2003
; Kamanga-Sollo et al., 2003
; Muller et al., 2003
; Wharton et al., 2004
). The morphogenic nature of the TGF-β family agrees with our conclusion that, at specific concentrations, TGF-β1 is permissive or nonpermissive to productive myogenic regenerative responses (, and S1
). Consequently, pronounced down-modulation of TGF-β1 causes immune disorders, inflammation and organ dysfunction. This complicates the use of TGF-β1 attenuators, such as antibodies and small molecules (delivered by pumps, or otherwise), or the use of genetically altered mice, which do not survive into old age if they harbor TGF-β pathway insufficiencies. For example, genetic deficiencies in TGF-β1,2,3, TGF-β RI,II,III, Smad2, Smad4 and others (Oshima et al., 1996
; Bonyadi et al., 1997
; Larsson et al., 2001
; Brionne et al., 2003
; Andrews et al., 2006
) all cause embryonic or neonatal lethality, due to the abnormal development of various organ systems. Furthermore, Smad3 knockout animals survive after birth, but die before old age due to chronic infections, accelerated rate of cancers and T-cell dysfunction (Datto et al., 1999
; Yang et al., 1999
). TGF-β1 heterozygous mice also survive after birth, but have increased neuronal abnormalities, inflammation and fibrosis of blood vessels (Wyss-Coray et al., 1997, 2001
; Brionne et al., 2003
). Combined, these data agree with our conclusion that prolonged, or sustained, experimental down-modulation of TGF-β1 levels will perturb cellular function.
Our work reveals that platelet-derived sera TGF-β1 levels, or endocrine TGF-β1 levels, do not explain the age-dependent inhibition of tissue regeneration by this cytokine. Specifically, if all endogenous young sera TGF-β1 were active, it would readily suppress satellite cell responses, in vitro
( and ). Further, neutralization of circulatory TGF-β1 is not achievable by experimental ligand traps, in vivo
( and ). These data suggest the presence of a natural TGF-β1 modifier/decoy (predictably more active in the young), which is likely endocrine in nature, or released during platelet activation ( and ). Future work on the characterization of such natural systemic antagonist(s) of TGF-β1 may also explain the regenerative phenotypes observed in hetereochronic parabiosis (Conboy et al., 2005
This work further confirms and extrapolates the autocrine/paracrine mode of TGF-β age-dependent inhibition of organ stem cell responses (Carlson et al., 2008
). Specifically, old tissue repair was improved only when P-Smad3 levels were attenuated locally in muscle (e.g. through small molecule systemic delivery), which is also expected to attenuate TGF-β signaling broadly in all tissues ( and ). In contrast, systemically administered neutralizing antibody, or soluble receptor, failed to restore old muscle repair. These molecules did not attenuate endocrine or local TGF-β1, nor diminish P-Smad3 levels in satellite cells, in vivo
( and ). These data also predict the presence of a positive feed-back loop, where a certain TGF-β signaling strength is required to maintain TGF-β1 ligand expression (), which was additionally suggested by TGF-β level reduction in muscle treated with RNAi to Smad3 (Carlson et al., 2008
). It remains to be determined whether there is a broad TGF-β1 increase within old tissues (in addition to skeletal muscle fibers), or if a narrow subset of cells in aged organisms overproduce TGF-β1.
While the involvement of additional TGF-β family members (or other age-dependent cytokines involved with muscle repair decline) is certainly not ruled out, TGF-β1 was identified here as a key, age-related inhibitor of myogenic responses. Additionally, myostatin was previously reported to not locally increase in old muscle tissue (Carlson et al., 2008
), thus implicating the ubiquitous TGF-β1, rather than tissue-specific family members in these age-related phenotypes. While other circulatory molecules may contribute to the aging of muscle stem cell responses, our data suggest that Wnt is not a systemic age-dependent attenuator of myogenicity (, S2
). These data are in apparent disagreement with those reported by Brack et al. (2008)
; and further study will resolve whether Wnt is present in the circulation, is upregulated with aging and becomes inhibitory to myogenic responses at some age-specific level. Of note, in reports examining Wnt in young mice only, it is suggested that Wnt promotes satellite cell myogenicity for both differentiation of myoblasts to myotubes, and with respect to satellite cell self-renewal (Brack et al., 2008
; Le Grand et al., 2009
Comprehensively, these studies identify physiological sources of TGF-β1 that are responsible for age-related muscle repair inhibition, and point toward novel therapeutic strategies for the enhancement of organ regenerative potential. In proof of principle research, intra-muscular attenuation of Smad3, by RNAi, rescued aged muscle repair (Carlson et al., 2008
). Such methodology, however, is not easily applicable to an organism-wide restoration of old organ repair. In contrast, our herein reported systemic delivery of a TGF-β receptor kinase inhibitor allows one to reach all tissues, to recalibrate TGF-β1 ligand levels to their youthful states and to potentially rejuvenate their regenerative function.