Overexpression of cyclin E/CDK2 complexes overcomes FGF-induced cell cycle arrest in the presence of hypophosphorylated Rb proteins

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.     

Overexpression of cyclin E/CDK2 complexes overcomes FGF-induced cell cycle arrest in the presence of hypophosphorylated Rb proteins

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.     

Downregulation of Akt activity contributes to the growth arrest induced by FGF in chondrocytes

Unregulated FGF signaling produced by activating FGFR3 mutations causes several forms of dwarfism-associated chondrodysplasias in humans and mice. FGF signaling inhibits chondrocyte proliferation by activating multiple signal transduction pathways that all contribute to chondrocyte growth arrest and induction of some aspects of differentiation. Previous studies had identified the Stat1 pathway, dephosphorylation of the Rb family proteins p107 and p130, induction of p21 expression and sustained activation of MAP kinases as playing a role in the FGF response of chondrocytes. We have examined the role of Akt (PKB) in the response of chondrocytes to FGF signaling. Differently from what is observed in many other cell types, FGF does not activate Akt in chondrocytes, and Akt phosphorylation is actually downregulated after FGF treatment. By expressing a constitutively activated, myristylated form of Akt (myr-Akt) in the RCS chondrosarcoma cell line, we show that Akt activation partially counteracts the inhibitory effect of FGF signaling. The response of myr-Akt expressing cells to FGF is identical to parental RCS in the first few hours after treatment, but then diverges as myr-Akt cells show decreased p130 phosphorylation, increased cyclin E/cdk2 activity and continue to proliferate at a slow rate. Constitutive Akt activation does not affect p21 expression but appears to influence directly cdk/cyclin activity. On the other hand, the induction of differentiation-related genes is unchanged in myr-Akt cells. These results identify Akt downregulation as an important aspect of the response of chondrocytes to FGF that, however, only affects chondrocyte proliferation and not the ability of FGF to induce differentiation genes.     

Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes

Fibroblast growth factors (FGFs) regulate long bone development by affecting the proliferation and differentiation of chondrocytes. FGF treatment inhibits the proliferation of chondrocytes both in vitro and in vivo, but the signaling pathways involved have not been clearly identified. In this report we show that both the MEK-ERK1/2 and p38 MAPK pathways, but not phospholipase C gamma or phosphatidylinositol 3-kinase, play a role in FGF-mediated growth arrest of chondrocytes. Chemical inhibitors of the MEK1/2 or the p38 MAPK pathways applied to rat chondrosarcoma (RCS) chondrocytes significantly prevented FGF-induced growth arrest. The retinoblastoma family members p107 and p130 were previously shown to be essential effectors of FGF-induced growth arrest in chondrocytes. The dephosphorylation of p107, one of the earliest events in RCS growth arrest, was significantly blocked by MEK1/2 inhibitors but not by the p38 MAPK inhibitors, whereas that of p130, which occurs later, was partially prevented both by the MEK and p38 inhibitors. Furthermore, by expressing the nerve growth factor (NGF) receptor, TrkA, and the epidermal growth factor (EGF) receptor, ErbB1, in RCS cells we show that NGF treatment of the transfected cells caused growth inhibition, whereas EGF did not. FGF- and NGF-induced growth inhibition is accompanied by a strong and sustained activation of ERK1/2 and p38 MAPK and a decrease of AKT phosphorylation, whereas EGF induces a much more transient activation of p38 and ERK1/2 and increases AKT phosphorylation. These results indicate that inhibition of chondrocyte proliferation by FGF requires both ERK1/2 and p38 MAPK signaling and also suggest that sustained activation of these pathways is required to achieve growth inhibition.     

The B55α Regulatory Subunit of Protein Phosphatase 2A Mediates Fibroblast Growth Factor-Induced p107 Dephosphorylation and Growth Arrest in Chondrocytes

Fibroblast growth factor (FGF)-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types and underlies several genetic skeletal disorders caused by activating FGF receptor (FGFR) mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the retinoblastoma protein (Rb) family member p107 by protein phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report, we show that the PP2A-B55α holoenzyme (PP2A containing the B55α subunit) is responsible for this phenomenon. Only the B55α (55-kDa regulatory subunit, alpha isoform) regulatory subunit of PP2A was able to bind p107, and this interaction was induced by FGF in chondrocytes but not in other cell types. Small interfering RNA (siRNA)-mediated knockdown of B55α prevented p107 dephosphorylation and FGF-induced growth arrest of RCS (rat chondrosarcoma) chondrocytes. Importantly, the B55α subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55α is also the site of cyclin-dependent kinase (CDK) binding, B55α association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55α subunit itself on several serine residues that drastically increases the affinity of B55α for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55α dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals.     

Activation of p107 by Fibroblast Growth Factor,Which Is Essential for Chondrocyte Cell Cycle Exit,Is Mediated by the Protein Phosphatase 2A/B55α Holoenzyme

The phosphorylation state of pocket proteins during the cell cycle is determined at least in part by an equilibrium between inducible cyclin-dependent kinases (CDKs) and serine/threonine protein phosphatase 2A (PP2A). Two trimeric holoenzymes consisting of the core PP2A catalytic/scaffold dimer and either the B55α or PR70 regulatory subunit have been implicated in the activation of p107/p130 and pRB, respectively. While the phosphorylation state of p107 is very sensitive to forced changes of B55α levels in human cell lines, regulation of p107 in response to physiological modulation of PP2A/B55α has not been elucidated. Here we show that fibroblast growth factor 1 (FGF1), which induces maturation and cell cycle exit in chondrocytes, triggers rapid accumulation of p107-PP2A/B55α complexes coinciding with p107 dephosphorylation. Reciprocal solution-based mass spectrometric analysis identified the PP2A/B55α complex as a major component in p107 complexes, which also contain E2F/DPs, DREAM subunits, and/or cyclin/CDK complexes. Of note, p107 is one of the preferred partners of B55α, which also associates with pRB in RCS cells. FGF1-induced dephosphorylation of p107 results in its rapid accumulation in the nucleus and formation of larger complexes containing p107 and enhances its interaction with E2F4 and other p107 partners. Consistent with a key role of B55α in the rapid activation of p107 in chondrocytes, limited ectopic expression of B55α results in marked dephosphorylation of p107 while B55α knockdown results in hyperphosphorylation. More importantly, knockdown of B55α dramatically delays FGF1-induced dephosphorylation of p107 and slows down cell cycle exit. Moreover, dephosphorylation of p107 in response to FGF1 treatment results in early recruitment of p107 to the MYC promoter, an FGF1/E2F-regulated gene. Our results suggest a model in which FGF1 mediates rapid dephosphorylation and activation of p107 independently of the CDK activities that maintain p130 and pRB hyperphosphorylation for several hours after p107 dephosphorylation in maturing chondrocytes.     

A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation

Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E-Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate.     

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G2 arrest in RCS chondrocytes

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G1 phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G2 phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G1 arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. The inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G2 arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and ATM/ATR kinase are known to play essential roles in the G2 checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G2 arrest. Additionally our results indicate that the transient G2 arrest is induced by FGF in RCS cell through mechanisms that are independent of the G1 arrest, and that the G2 block is not strictly required for the sustained G1 arrest but may provide a pausing mechanism that allows the FGF response to be fully established.     

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G2 arrest in RCS chondrocytes

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G₁ phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G₂ phase of the cycle for the first 4–6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G₁ arrest.The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase. That, however, appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop and not the activation of specific phosphatases. The inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling and not a consequence of the G₂ arrest as can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and ATM/ATR kinase are known to play essential roles in the G₂ checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G₂ arrest.Additionally, our results indicate that the transient G₂ arrest is induced by FGF in RCS cell through mechanisms that are independent of the G₁ arrest, and that the G₂ block is not strictly required for the sustained G₁ arrest but may provide a pausing mechanism that allows the FGF response to be fully established.Key words: fibroblast growth factor, chondrocyte, G₂/M arrest, Myt1, cyclin B1, CDK1     

FGF signaling targets the pRb-related p107 and p130 proteins to induce chondrocyte growth arrest

Unregulated FGF signaling affects endochondral ossification and long bone growth, causing several genetic forms of human dwarfism. One major mechanism by which FGFs regulate endochondral bone growth is through their inhibitory effect on chondrocyte proliferation. Because mice with targeted mutations of the retinoblastoma (Rb)-related proteins p107 and p130 present severe endochondral bone defects with excessive chondrocyte proliferation, we have investigated the role of the Rb family of cell cycle regulators in the FGF response. Using a chondrocyte cell line, we found that FGF induced a rapid dephosphorylation of all three proteins of the Rb family (pRb, p107, and p130) and a blockade of the cells in the G1 phase of the cell cycle. This cell cycle block was reversed by inactivation of Rb proteins with viral oncoproteins such as polyoma large T (PyLT) antigen and Adenovirus E1A. Expression of a PyLT mutant that efficiently binds pRb, but not p107 and p130, allowed the cells to be growth inhibited by FGF, suggesting that pRb itself is not involved in the FGF response. To investigate more precisely the role of the individual Rb family proteins in FGF-mediated growth inhibition, we used chondrocyte micromass culture of limb bud cells isolated from mice lacking Rb proteins individually or in combination. Although wild-type as well as Rb-/- chondrocytes were similarly growth inhibited by FGF, chondrocytes null for p107 and p130 did not respond to FGF. Furthermore, FGF treatment of metatarsal bone rudiments obtained from p107-/-;p130-/- embryos failed to inhibit proliferation of growth plate chondrocytes, whereas rudiments from p107-null or p130-null embryos showed only a slight inhibition of growth. Our findings indicate that p107 and p130, but not pRb, are critical effectors of FGF-mediated growth inhibition in chondrocytes.     

Oxidative stress induces protein phosphatase 2A-dependent dephosphorylation of the pocket proteins pRb,p107, and p130

Oxidative stress induces cell death and growth arrest. In this study, the regulation and the functional role of the retinoblastoma family proteins pRb, p107, and p130 in the cellular response to oxidative stress were investigated. Treatment of endothelial cells with H2O2 induced rapid hypophosphorylation of the retinoblastoma family proteins. This event did not require p53 or p21Waf1/Cip1/Sdi1 and was not associated with cyclin/cyclin-dependent kinase down-modulation. Four lines of evidence indicate that H2O2-induced hypophosphorylation of pRb, p107, and p130 was because of the activity of protein phosphatase 2A (PP2A). First, cell treatment with two phosphatase inhibitors, okadaic acid and calyculin A, prevented the hypophosphorylation of the retinoblastoma family proteins, at concentrations that specifically inhibit PP2A. Second, SV40 small t, which binds and inhibits PP2A, when overexpressed prevented H2O2-induced dephosphorylation of the retinoblastoma family proteins, whereas a SV40 small t mutant unable to bind PP2A was totally inert. Third, PP2A core enzyme physically interacted with pRb and p107, both in H2O2-treated and untreated cells. Fourth, a PP2A phosphatase activity was co-immunoprecipitated with pRb, and the activity of pRb-associated PP2A was positively modulated by cell treatment with H2O2. Because DNA damaging agents inhibit DNA synthesis in a pRb-dependent manner, it was determined whether the PP2A-mediated dephosphorylation of the retinoblastoma family proteins played a role in this S-phase response. Indeed, it was found that inhibition of PP2A by SV40 small t over-expression prevented DNA synthesis inhibition induced by H2O2.     

p21CIP1 and Cdc25A: competition between an inhibitor and an activator of cyclin-dependent kinases.

Cdc25A, a phosphatase essential for G1-S transition, associates with, dephosphorylates, and activates the cell cycle kinase cyclin E-cdk2. p21CIP1 and p27 are cyclin-dependent kinase (cdk) inhibitors induced by growth-suppressive signals such as p53 and transforming growth factor beta (TGF-beta). We have identified a cyclin binding motif near the N terminus of Cdc25A that is similar to the cyclin binding Cy (or RR LFG) motif of the p21CIP1 family of cdk inhibitors and separate from the catalytic domain. Mutations in this motif disrupt the association of Cdc25A with cyclin E- or cyclin A-cdk2 in vitro and in vivo and selectively interfere with the dephosphorylation of cyclin E-cdk2. A peptide based on the Cy motif of p21 competitively disrupts the association of Cdc25A with cyclin-cdks and inhibits the dephosphorylation of the kinase. p21 inhibits Cdc25A-cyclin-cdk2 association and the dephosphorylation of cdk2. Conversely, Cdc25A, which is itself an oncogene up-regulated by the Myc oncogene, associates with cyclin-cdk and protects it from inhibition by p21. Cdc25A also protects DNA replication in Xenopus egg extracts from inhibition by p21. These results describe a mechanism by which the Myc- or Cdc25A-induced oncogenic and p53- or TGF-beta-induced growth-suppressive pathways counterbalance each other by competing for cyclin-cdks.     

Altered regulation of cell cycle machinery involved in interleukin-1-induced G(1) and G(2) phase growth arrest of A375S2 human melanoma cells

Interleukin-1 (IL-1) inhibits the growth of A375S2 human melanoma cells by arresting them at G(1) and G(2) phases of the cell cycle. The arrests are preceded by a rapid decrease in kinase activities of cyclin E-Cdk2 and cyclin B1-Cdc2, which are critical for G(1)-S and G(2)-M progression, respectively. IL-1 quickly enhances the protein expression of the CDK inhibitor p21(cip1). The induced p21 binds preferentially to cyclin E-Cdk2, and the increase in p21 binding parallels the decrease in cyclin E-Cdk2 activity. Thus, p21 is likely to be responsible for the inhibition of cyclin E-Cdk2 activity and G(1) arrest. Coinciding with the decrease in cyclin B1-Cdc2 activity, there is an increase in tyrosine phosphorylation of Cdc2, suggesting that an increase in the inactive Tyr-15-phosphorylated form of Cdc2 is involved in the decrease in cyclin B1-Cdc2 activity and G(2) arrest. Furthermore, we found that IL-1 causes rapid dephosphorylation of p107, but not of pRb or p130, while the total protein levels of p130 are increased. Thus, IL-1 may exert its growth-arresting effects via p107 and p130 pathways rather than through pRb.     

Fibroblast growth factor inhibits chondrocytic growth through induction of p21 and subsequent inactivation of cyclin E-Cdk2

Fibroblast growth factor (FGF) and its receptor (FGFR) are thought to be negative regulators of chondrocytic growth, as exemplified by achondroplasia and related chondrodysplasias, which are caused by constitutively active mutations in FGFR3. To understand the growth-inhibitory mechanisms of FGF, we analyzed the effects of FGF2 on cell cycle-regulating molecules in chondrocytes. FGF2 dramatically inhibited proliferation of rat chondrosarcoma (RCS) cells and arrested their cell cycle at the G(1) phase. FGF2 increased p21 expression in RCS cells, which assembled with the cyclin E-Cdk2 complexes, although the expression of neither cyclin E nor Cdk2 increased. In addition, the kinase activity of immunoprecipitated cyclin E or Cdk2, assessed with retinoblastoma protein (pRb) as substrate, was dramatically reduced by FGF-2. Moreover, FGF2 shifted pRb to its underphosphorylated, active form in RCS cells. FGF2 not only induced p21 protein expression in proliferating chondrocytes in mouse fetal limbs cultured in vitro but also decreased their proliferation as assessed by the expression of histone H4 mRNA, a marker for cells in S phase. Furthermore, inhibitory effects of FGF2 on chondrocytic proliferation were partially reduced in p21-null limbs, compared with those in wild-type limbs in vitro. Taken together, FGF's growth inhibitory effects of chondrocytes appear to be mediated at least partially through p21 induction and the subsequent inactivation of cyclin E-Cdk2 and activation of pRb.     

Cdk7- and Cdc25A-independent dephosphorylation of Cdk2 during phorbol ester-mediated cell cycle arrest in U937 cells

The molecular mechanism underlying protein kinase C (PKC)-mediated cell cycle arrest is poorly understood. We undertook to characterize phorbol ester-activated PKC-mediated cell cycle arrest. Treatment with phorbol ester inhibited cell growth of human histiocytic lymphoma U937 cells with 83% of the cells arrested in G1 phase. Reduced activity of cdk2 correlated with cdk2 dephosphorylation and accumulation of cdk2 inhibitor p21Waf in phorbol ester-treated cells. Dephosphorylation of cdk2 was not associated with cdk7 and cdc25A activity in phorbol ester-treated cells. Protein phosphatase inhibitor assays suggest that the dephosphorylation of cdk2 results in the activation of a specific protein tyrosine phosphatase. Thus, dephosphorylation of cdk2 as well as accumulation of cdk2 inhibitor is likely to contribute to the G1 phase arrest in phorbol ester-treated in U937 cells.     

Progesterone inhibits estrogen-induced cyclin D1 and cdk4 nuclear translocation, cyclin E- and cyclin A-cdk2 kinase activation, and cell proliferation in uterine epithelial cells in mice

The response of the uterine epithelium to female sex steroid hormones provides an excellent model to study cell proliferation in vivo since both stimulation and inhibition of cell proliferation can be studied. Thus, when administered to ovariectomized adult mice 17beta-estradiol (E2) stimulates a synchronized wave of DNA synthesis and cell division in the epithelial cells, while pretreatment with progesterone (P4) completely inhibits this E2-induced cell proliferation. Using a simple method to isolate the uterine epithelium with high purity, we have shown that E2 treatment induces a relocalization of cyclin D1 and, to a lesser extent, cdk4 from the cytoplasm into the nucleus and results in the orderly activation of cyclin E- and cyclin A-cdk2 kinases and hyperphosphorylation of pRb and p107. P4 pretreatment did not alter overall levels of cyclin D1, cdk4, or cdk6 nor their associated kinase activities but instead inhibited the E2-induced nuclear localization of cyclin D1 to below the control level and, to a lesser extent, nuclear cdk4 levels, with a consequent inhibition of pRb and p107 phosphorylation. In addition, it abrogated E2-induced cyclin E-cdk2 activation by dephosphorylation of cdk2, followed by inhibition of cyclin A expression and consequently of cyclin A-cdk2 kinase activity and further inhibition of phosphorylation of pRb and p107. P4 is used therapeutically to oppose the effect of E2 during hormone replacement therapy and in the treatment of uterine adenocarcinoma. This study showing a novel mechanism of cell cycle inhibition by P4 may provide the basis for the development of new antiestrogens.     

Bisindolylmaleimide I suppresses fibroblast growth factor-mediated activation of Erk MAP kinase in chondrocytes by preventing Shp2 association with the Frs2 and Gab1 adaptor proteins

Fibroblast growth factors (FGFs) inhibit chondrocyte proliferation via the Erk MAP kinase pathway. Here, we explored the role of protein kinase C in FGF signaling in chondrocytes. Erk activity in FGF2-treated RCS (rat chondrosarcoma) chondrocytes or human primary chondrocytes was abolished by the protein kinase C inhibitor bisindolylmaleimide I (Bis I). Bis I inhibited FGF2-induced activation of MEK, Raf-1, and Ras members of Erk signaling module but not the FGF2-induced tyrosine phosphorylation of Frs2 or the kinase activity of FGFR3, demonstrating that it targets the Erk cascade immediately upstream of Ras. Indeed, Bis I abolished the FGF2-mediated association of Shp2 tyrosine phosphatase with Frs2 and Gab1 adaptor proteins necessary for proper Ras activation. We also determined which PKC isoform is involved in FGF2-mediated activation of Erk. When both conventional and novel PKCs expressed by RCS chondrocytes (PKCalpha, -gamma, -delta, and -epsilon) were down-regulated by phorbol ester, cells remained responsive to FGF2 with Erk activation, and this activation was sensitive to Bis I. Moreover, treatment with PKClambda/zeta pseudosubstrate lead to significant reduction of FGF2-mediated activation of Erk, suggesting involvement of an atypical PKC.     

Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors.

Understanding how cyclin-cdk complexes recognize their substrates is a central problem in cell cycle biology. We identified an E2F1-derived eight-residue peptide which blocked the binding of cyclin A and E-cdk2 complexes to E2F1 and p21. Short peptides spanning similar sequences in p107, p130, and p21-like cdk inhibitors likewise bound to cyclin A-cdk2 and cyclin E-cdk2. In addition, these peptides promoted formation of stable cyclin A-cdk2 complexes in vitro but inhibited the phosphorylation of the retinoblastoma protein by cyclin A- but not cyclin B-associated kinases. Mutation of the cyclin-cdk2 binding motifs in p107 and E2F1 likewise prevented their phosphorylation by cyclin A-associated kinases in vitro. The cdk inhibitor p21 was found to contain two functional copies of this recognition motif, as determined by in vitro kinase binding/inhibition assays and in vivo growth suppression assays. Thus, these studies have identified a cyclin A- and E-cdk2 substrate recognition motif. Furthermore, these data suggest that p21-like cdk inhibitors function, at least in part, by blocking the interaction of substrates with cyclin-cdk2 complexes.     

p107 and p130 Coordinately regulate proliferation,Cbfa1 expression,and hypertrophic differentiation during endochondral bone development

During endochondral bone development, both the chondrogenic differentiation of mesenchyme and the hypertrophic differentiation of chondrocytes coincide with the proliferative arrest of the differentiating cells. However, the mechanisms by which differentiation is coordinated with cell cycle withdrawal, and the importance of this coordination for skeletal development, have not been defined. Through analysis of mice lacking the pRB-related p107 and p130 proteins, we found that p107 was required in prechondrogenic condensations for cell cycle withdrawal and for quantitatively normal alpha1(II) collagen expression. Remarkably, the p107-dependent proliferative arrest of mesenchymal cells was not needed for qualitative changes that are associated with chondrogenic differentiation, including production of Alcian blue-staining matrix and expression of the collagen IIB isoform. In chondrocytes, both p107 and p130 contributed to cell cycle exit, and p107 and p130 loss was accompanied by deregulated proliferation, reduced expression of Cbfa1, and reduced expression of Cbfa1-dependent genes that are associated with hypertrophic differentiation. Moreover, Cbfa1 was detected, and hypertrophic differentiation occurred, only in chondrocytes that had undergone or were undergoing a proliferative arrest. The results suggest that Cbfa1 links a p107- and p130-mediated cell cycle arrest to chondrocyte terminal differentiation.