There is growing awareness of the pleiotropic effects of Pb, with targets of toxicity including multiple tissues that include the skeleton. In fact, at concentrations considered subtoxic, Pb is known to affect skeletal growth. The two most recent National Health and Nutrition Examination Surveys (NHANES II and NHANES III) have demonstrated that Pb exposure reduces skeletal growth independent of factors such as nutritional status, social/ economic class, and co-morbid diseases (Ballew et al. 1999
). Moreover, significant developmental effects are apparent at blood concentrations as low as 4 μg/dl (Ballew et al. 1999
). Thus, skeletal tissues, and particularly the growth plate, are among the most sensitive tissue targets of Pb. Representing a first step toward understanding the mechanism underlying the influence of Pb on the skeleton, findings presented in this article demonstrate that Pb targets mesenchymal stem cells and affects the determination of cell fate. Specifically, embryonic limb bud cells exposed to Pb display a severalfold increase in chondrogenenic commitment. Moreover, Pb induces the chondrogenic effects of both BMP-2 and TGF-β. Because embryonic limb bud stem cells also have the potential to differentiate into other mesenchymal tissues, Pb possibly also influences the cell fate on other cell differentiation pathways.
Pb has broad and complex effects on multiple intracellular signaling pathways. As mentioned above, Pb has been shown in various tissues to a
) block calcium signaling and inhibit Ca2+
/phospholipid-dependent PKC signaling (Pokorski et al. 1999
; Vazquez and Pena 2004
) induce ERK1/2 and p38 (MAPK) phosphorylation and activation (Lin et al. 2003
; Zhang et al. 2003
); and c
) activate AP-1 signaling (Hossain et al. 2000
). We have found that Pb affects neither basal BMP-2 nor TGF-β signaling; thus it is unlikely that the effect of Pb on chondrogenesis is related to a direct alteration of these signaling pathways. Moreover, although Pb enhanced the effect of BMP-2 on chondrogenesis, it acted as an inhibitor of Smad1/5/8 signaling. Further experiments demonstrated that Pb also regulates other signaling pathways, including AP-1 and possibly NFκB. Thus, it appears that the effects of induction of chondrogenesis and enhancement of TGF-β and BMP-2 on chondrogenesis are secondary to stimulation of other interacting signaling pathways.
Previous work has established that Pb regulates BMP and TGF-β signaling pathways. Oral administration of Pb chloride has been shown to cause a reduction in the expression of TGF-β in intestinal tissues in diabetes-prone NOD mice (Goebel et al. 1999
). In an earlier study using chicken growth plate chondrocyte cultures (Zuscik et al. 2002b
), we found that both Pb and TGF-β independently inhibit expression of the maturational marker colX
, Interestingly, this study also showed that inhibition of colX
by TGF-β was completely reversed by Pb. Furthermore, although neither Pb nor TGF-β alone affected the expression of BMP-6, in combination they induced its expression 3-fold (Zuscik et al. 2002b
). Although these previous studies did not fully examine regulation of downstream signaling events, they did demonstrate a regulation of TGF-β signaling. When considered along with the current findings, it seems clear that the influence of Pb on chondrogenesis/chondrocyte differentiation is at least partially dependent upon the presence of TGF-β and activation of its Smad signaling pathway. Overall, our findings suggest that Pb exposure renders mesenchymal precursor cells more sensitive to TGF-β by increasing Smad3 phosphorylation and signaling. To our knowledge, this is the first demonstration of regulation of Smad phosphorylation by Pb in any cell system.
In the present study, we found that BMP signaling is also regulated by Pb. BMP receptor signaling occurs in a manner analagous to the TGF-β pathway, with Smad1/5/8 binding to the type I BMP receptor, followed by phosphorylation of these factors upon ligand activation (Miyazono et al. 2005
). Using a polyclonal antibody that recognizes all three BMP receptor–associated Smads, we found that the basal phosphorylation state was not altered by Pb alone, but the induction of phosphorylation of Smad1/5/8 was markedly inhibited by Pb. Similarly, although Pb did not alter basal activation of the BMP-Smad responsive reporter 12 × SBE, Pb significantly reduced activation of this reporter by BMP-2. Thus, similar to TGF-β signaling, Pb regulates BMP signaling only during activation of the pathway by lig-and. Interestingly, the effects are opposite, with Pb inhibiting BMP-Smad phosphorylation and stimulating TGF-β-Smad phosphorylation. Overall, the potent inhibition of BMP-2–induced Smad1/5/8 signaling by Pb represents the most robust signaling effect identified to date in a skeletal cell type with regard to a candidate mechanism of Pb toxicity.
The complex regulation of the Smad signaling molecules makes it unlikely that the induction of chondrogenesis by Pb is cause by direct alteration of these pathways. Although BMP and TGF-β signaling pathways have antagonistic effects on some cells, including growth plate chondrocytes where TGF-β inhibits and BMP-2 stimulates maturation, both signals enhance chondrogenesis in mesenchymal stem cell populations (Hoffmann and Gross 2001
). An earlier study has shown that BMP-4 stimulates chondrogenesis in C3H10T1/2 and MC615 chondroprogenitor cells through activation of the BMP-receptor–associated Smads (Hatakeyama et al. 2003
). TGF-β signaling through Smad2 and Smad3 is associated with enhanced chondrogenesis in murine mesenchymal stem cells (Yu and Xing 2006
). Additionally, the action of these factors is dependent on complex signaling, because it is clear that TGF-β and BMP signals act in combination with other signaling pathways. In ATDC5 cells, stimulation of chondrogenesis by TGF-β is mediated by activation of Smad pathways and the p38 and Erk1/2 MAP kinase pathways simultaneously (Watanabe et al. 2001
). Furthermore, whereas Wnt/β-catenin signaling in vivo
appears to stimulate osteogenesis over chondrogenesis (Day et al. 2005
), in human mesenchymal stem cell cultures TGF-β acts synergistically with Wnt/β-catenin signaling to induce chondrogenesis (Zhou et al. 2004
). Similarly, BMPs have been shown to act synergistically with Wnt/β-catenin signaling to induce chondrogenesis in C3H10T1/2 cells (Fischer et al. 2002
). Overall, it is clear that BMP and TGF-β Smad signaling is critical for induction of chondrogenesis; however, these pathways are only a part of multiple signaling events that contribute to the regulation of chondrogenic commitment.
To extend these signaling results, we also examined the effect of Pb on other signaling pathways. Although no effects were observed on CRE and TOPFLASH activation using the respective luciferase-based reporters, the experiments showed that Pb inhibited basal AP-1-Luc reporter activity. AP-1 activation has been shown to inhibit chondrogenesis, so it is possible that the inhibition of AP-1 could be involved in the induction of chondrogenesis by Pb (Hwang et al. 2005
; Tufan et al. 2002
). The inhibition of AP-1 signaling in mesenchymal stem cells is in contrast to the findings in other cells where Pb induces AP-1 signaling activity. In a previous study (Zuscik et al. 2002b
), we showed that Pb increased AP-1 and NFκB signaling in chick embryonic chondrocytes in culture. Furthermore, in vitro
Pb exposure in rats results in activation of AP-1 and NFκB levels in multiple regions of the brain and in astrocytes in culture (Ramesh et al. 2001
). In vivo
Pb exposure results in increased NFκB signaling in renal tubular cells and results in nephritis in rats (Rodriguez-Iturbe et al. 2005
). Similarly we found that Pb induces NFκB signaling in MSCs as measured by induction of NFκB-Luc reporter. However, because signaling on the NFκB pathway has been shown to destabilize Sox9 mRNA and inhibit chondrogenesis (Sitcheran et al. 2003
), it is unlikely that Pb-induction of NFκB signaling seen in MSCs is directly causing an enhanced chondrogenic response.
The induction of chondrogenesis by Pb in the current study is consistent with findings observed in an in vivo
murine model of fracture healing (Carmouche et al. 2005
). Mice with Pb levels similar to those found in humans with Pb intoxication had delayed healing of stabilized femur fractures. The effect was dose dependent, and cartilage was observed to be a major target. Pb-exposed mice had increased cartilage volumes, delayed chondrocyte maturation, persistence of cartilage, and reduced bone formation (Carmouche et al. 2005
). Overall, when considered along with the results of Carmouche et al. (2005)
, our findings in the present study further support the hypothesis that Pb is an inducer of chondrogenesis.
In conclusion, the present study establishes that in addition to affecting chondrocyte maturation, Pb accelerates the differentiation of mesenchymal precursors into chondrocytes. Although Pb alters BMP and TGF-β signaling, the mechanism through which Pb regulates mesenchymal cell fate determination is complex and likely involves modulation and integration of multiple signaling pathways. Increased understanding of the mechanisms through which Pb regulates stem cell fate and subsequently affects tissue repair is critical, given the sensitivity of these tissues and the ubiquitous presence of Pb in our society and in other industrialized and developing nations.