PTH effects on Mitogen-activated protein kinase (MAPK) pathways

MAPKs are serine-threonine protein kinases that are activated by diverse stimuli including cytokines, growth factors and hormones. MAPKs are activated by phosphorylation cascades. In fact, MAPK cascades consist of a core chain of three kinases, each of which is activated in turn through phosphorylation by the kinase positioned upstream of it. Thus, the MAPKs are phosphorylated and activated by the MAPK kinases (MAPKK, MKK or MEK), which are themselves activated by the MAPKK kinases (MAPKKK or MEKK) (Figure 6). The MAPKKKs are stimulated by diverse signals, including upstream proteins (such as Grb2, RAS) and G proteins (Gs, Gq, Gi), often derived from the activity of G-protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs).

The vast majority of defined substrates for MAPKs are transcription factors.

However, MAPKs have also the ability to phosphorylate many other substrates including other protein kinases, phospholipases, and cytoskeleton associated proteins. Therefore MAPK signaling pathways are involved in a diverse set of responses affecting cell fate, including cell proliferation and differentiation, adaptation to environmental stress and apoptosis (Figure 6) (208, 209).

As shown in Figure 6, there are mainly three families of mammalian MAPKs: the extracellular signal-regulated kinases1/2 (ERK1/2 or ERKs) which are also named MAPK; the c-Jun amino-terminal kinases1-3 (JNKs) and the p38(,,,) MAPKs.

Activation of the ERK1/2 cascade is important for proliferation, development and differentiation, whereas activation of JNK and p38 cascades are important for development, inflammation and apoptosis. This dichotomy, however, is an oversimplification, and the actual roles of each MAPK cascade are highly cell type and context dependent.

47

Figure 6. MAPKs signaling cascades in yeast and mammalian cells. See text for details. Adapted from Pierce et al., Nature Reviews (2002).

3.3.1 PTH1R-mediated MAPKs activation via Gs/cAMP/PKA and Gq/PLC/PKC signaling pathway

3.3.1.1 PTH effects on ERK1/2 pathway

PTH-induced ERK1/2 regulation has been described in various target cells, including osteoblastic cells, bone marrow cells and distal/proximal convoluted tubule kidney cells. In all these various cell types, ERK1/2 activation is associated with proliferation, differentiation, survival, and calcium transport (128, 129, 205, 206, 210-212).

Molecular mechanisms, by which ERK1/2 is regulated by PTH, are largely dependent on the cell type and experimental conditions. It has been demonstrated that PTH activates ERK1/2 mostly through a PKC-dependent mechanism (in osteosarcoma cells (UMR-106), rat primary osteoblasts, osteogenic cells from rat bone marrow precursors and in opossum kidney cells) (128, 205-207) but also through a 48

cAMP/PKA-dependent mechanism (in opossum kidney cells) (206). Interestingly, cAMP may have a dual function on ERKs activation depending on the presence or absence of specific kinases. Indeed, PTH-induced cAMP accumulation activates ERK1/2 and cell proliferation in cells expressing the MEK Kinase B-Raf, such as in murine osteoblasts (MC3T3-MC4) and chondrocytes (ATDC5), but inhibits growth in B-Raf-lacking cells, such as in osteosarcoma cells (MG63). Moreover, all these effects were found to be PKA-independent (129). Thus, in B-Raf-expressing cells, PTH-induced cAMP production activates ERK1/2 through the Epac/Rap1/B-Raf signaling pathway (Figure 7), whereas in B-Raf-lacking cells, cAMP suppresses ERK1/2 activation.

Moreover, PTH-induced cAMP accumulation attenuates ERK1/2 activation induced by other growth factors such as EGF (Epidermal Growth Factor) and FGF (Fibroblast Growth Factor) (126, 211). This attenuation of ERK1/2 phosphorylation was found to be PKA-dependent (126) and is correlated with anti-proliferative actions (126, 211).

In summary, PTH may activate ERK1/2 and osteoblastic proliferation via various signaling pathways. Generally osteoblastic proliferation seems to be dependent on the Gq/PKC/ERK1/2 signaling cascade. On the other side, PTH-induced cAMP accumulation suppresses ERK1/2 activation (except in B-Raf expressing cells) and inhibits ERK1/2 activation induced by others growth factors such as EGF, PDGF and IGF-1. Since cAMP is crucial for PTH anabolic effects in vivo, one could speculate that ERK1/2 activation in vivo is therefore not involved in PTH anabolic effects. This hypothesis would also be consistent with the possibility that PTH increases osteoblast differentiation by suppressing proliferation (see point 2.2).

Figure 7 depicts multiple signaling pathways linking G-protein-coupled receptors (such as PTH1R) to the activation of ERK. It also shows that the signaling pathway linking GPCRs to ERK converges with that used by receptor tyrosine kinases (RTKs) at the level of Ras. Details are described in the legend of Figure 7.

49

MAPKKK

MAPKK

MAPK

Figure 7. Multiple pathways linking GPCRs to ERK (MAPK). Biochemical routes initiated by -subunits can stimulate Ras by the activation of receptor and non-receptor tyrosine kinases, which results in the recruitment of Sos to the membrane and the exchange of GDP for GTP bound to Ras. Activated Gq can stimulate Raf1 through protein kinase C (PKC) or by stimulating Ras by the Ca²⁺-dependent activation of RasGRF and tyrosine kinases acting on Sos. Gi, Go and Gs can also use tissue-restricted pathways regulating Rap, which can stimulate B-Raf and lead to the activation of MAPK. Activated MAPK translocates to the nucleus and phosphorylates nuclear proteins, including transcription factors, thereby regulating gene expression. Abbreviations: EPAC, exchange protein activated by cAMP; GAP, GTPase-activating protein; GRF, guanine-nucleotide releasing factor; MEK, MAPK kinase; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PLC-, phospholipase C.Adapted fromGutkind J.S., Science STKE (2000).

50

3.3.1.2 PTH effects on the JNK and p38 pathways

Little is known about JNK and p38 regulation by PTH. Nevertheless some studies suggest either their inhibition or activation depending on cell type.

Concerning the JNK pathway, in rat calvaria osteoblastic cells and in osteosarcoma cells (UMR-106), PTH-stimulated production cAMP enhances inhibition of c-Jun N-terminal kinase (JNK) in a PKA dependent manner (213, 214). More recently in rat enterocytes, PTH has been demonstrated to activate JNK by a Ca²⁺-dependent mechanism, which is linked to PTH regulation of intestinal cell proliferation (215).

Concerning the p38 pathway, several studies showed that PTH inhibits p38 activation in UMR-106 cells, primary human osteoblasts and primary bovine chondrocytes (216-218). On the other side, in UMR-106 cells, by using various specific inhibitors, Kwok et al. showed that PTH stimulation of LTBP-1 (latent transforming growth factor (TGF-)-binding protein) and TGF-1 mRNA expression was dependent on the PKA and the MAPK p38 and ERK1/2 pathways (219). In mouse primary osteoblastic cells, PTH stimulation of cox-2 mRNA was dependent on the PKA and the MAPK p38 pathway (219). Moreover, PTH may inhibit activation of p38 induced by various growth factors via a PKC-dependent and PKA-independent pathway. Thus, in chondrocytes, PTH-dependent inhibition of p38 was associated with retardation of cell hypertrophy, a differentiation process (220). Interestingly, in mouse primary osteoblasts, ALP production and mineral deposition was shown to be p38-dependent,. since inhibition of p38 with a pharmacological agent significantly affected osteoblast differentiation (221). In summary, PTH may inhibit p38, but in osteoblastic cells, basal activation of p38 seems important for PTH-induced osteoblastic cell differentiation.

51

4. Molecular mechanisms implicated in the regulation of