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.     

Pocket Protein p130/Rb2 Is Required for Efficient Herpes Simplex Virus Type 1 Gene Expression and Viral Replication

We have reported previously that herpes simplex virus type 1 (HSV-1) infection disrupts normal progression of the mammalian cell cycle, causing cells to enter a G(1)-like state. Infected cells were characterized by a decline in cyclin-dependent kinase 2 (CDK2) activities, loss of hyperphosphorylated retinoblastoma protein (pRb), accumulation of E2F-pocket protein complexes, and failure to initiate cellular DNA replication. In the present study, we investigated the role of the pocket proteins pRb, p107, and p130 in HSV-1-dependent cell cycle inhibition and cyclin kinase regulation by infecting murine 3T3 cells derived from wild-type (WT) mouse embryos or embryos with deletions of pRb (pRb(-/-)), p107 (p107(-/-)), p130 (p130(-/-)), or both p130 and p107 (p130(-/-)/p107(-/-)). With respect to CDK2 inhibition, viral protein accumulation, viral DNA replication, and progeny virus yield, WT, pRb(-/-), and p107(-/-) cells were essentially identical. In contrast, after infection of p130(-/-) cells, we observed no inhibition of CDK2 activity, a 5- to 6-h delay in accumulation of viral proteins, an impaired ability to form viral DNA replication compartments, and reduced viral DNA synthesis. As a result, progeny virus yield was reduced 2 logs compared to that in WT cells. Notably, p130(-/-)/p107(-/-) double-knockout cells had a virus replication phenotype intermediate between those of the p107(-/-) and p130(-/-) cells. We conclude from these studies that p130 is a key factor in regulating aspects of cell cycle progression, as well as the timely expression of viral genes and replication of viral DNA.     

Retinoblastoma protein (pRb), but not p107 or p130, is required for maintenance of enterocyte quiescence and differentiation in small intestine

The function of retinoblastoma protein (pRb) in the regulation of small intestine epithelial cell homeostasis has been challenged by several groups using various promoter-based Cre transgenic mouse lines. Interestingly, different pRb deletion systems yield dramatically disparate small intestinal phenotypes. These findings confound the function of pRb in this dynamic tissue. In this study, Villin-Cre transgenic mice were crossed with Rb (flox/flox) mice to conditionally delete pRb protein in small intestine enterocytes. We discovered a novel hyperplasia phenotype as well as ectopic cell cycle reentry within villus enterocytes in the small intestine. This phenotype was not seen in other pRb family member (p107 or p130) null mice. Using a newly developed crypt/villus isolation method, we uncovered that expression of pRb was undetectable, whereas proliferating cell nuclear antigen, p107, cyclin E, cyclin D3, Cdk2, and Cdc2 were dramatically increased in pRb-deficient villus cells. Cyclin A, cyclin D1, cyclin D2, and Cdk4/6 expression was not affected by absent pRb expression. pRb-deficient villus cells appeared capable of progressing to mitosis but with higher rates of apoptosis. However, the cycling villus enterocytes were not completely differentiated as gauged by significant reduction of intestinal fatty acid-binding protein expression. In summary, pRb, but not p107 or p130, is required for maintaining the postmitotic villus cell in quiescence, governing the expression of cell cycle regulatory proteins, and completing of absorptive enterocyte differentiation in the small intestine.     

The specific role of pRb in p16INK4A-mediated arrest of normal and malignant human breast cells

RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G₁ was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.Key words: breast cancer, senescence, retinoblastoma, p130, p107     

The specific role of pRb in p16INK4A-mediated arrest of normal and malignant human breast cells

RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G₁ was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.     

Vascular injury response in mice is dependent on genetic background

Mouse models are employed to unravel the pathophysiology of vascular restenosis. Although much effort has been spent on how to apply an adequate arterial injury, the influence of the genetic background of mice has not yet received sufficient consideration. The study presented herein was designed to demonstrate the influence of the mouse strain on vascular injury response. Mice of a defined background (50% 129 strain and 50% DBA strain) were backcrossed into either the 129 strain or the DBA strain. Male offspring were subjected to a femoral artery injury model by applying an electric current. Morphometric analysis revealed that backcrossing into the 129 strain resulted in a significant (P < 0.001) 17-fold increase in neointima formation (n = 17 mice) compared with backcrossing into the DBA strain (n = 19). The values of neointima area were 9.18 x 10(3) +/- 2.13 x 10(3) and 0.54 x 10(3) +/- 0.39 x 10(3) microm2, respectively. In conjunction, the vessel wall area was enhanced by 1.8-fold (P < 0.001). In contrast, no significant differences were found for the areas of the lumen and the tunica media. Similarly, a significant increase in neointima formation was also found for mice of pure 129 strain compared with pure DBA strain. The results underline the importance of the genetic background for studies on vascular injury response. Furthermore, because the mouse genome of the various strains is well defined, serial testing of the genetic background of mice will provide candidate genes and/or genetic modifiers controlling vascular injury response.     

Unique and overlapping functions of pRb and p107 in the control of proliferation and differentiation in epidermis

The retinoblastoma gene product, pRb, plays a crucial role in cell cycle regulation, differentiation and inhibition of oncogenic transformation. pRb and its closely related family members p107 and p130 perform exclusive and overlapping functions during mouse development. The embryonic lethality of Rb-null animals restricts the phenotypic analysis of these mice to mid-gestation embryogenesis. We employed the Cre/loxP system to study the function of Rb in adult mouse stratified epithelium. Rb(F19/F19);K14cre mice displayed hyperplasia and hyperkeratosis in the epidermis with increased proliferation and aberrant expression of differentiation markers. In vitro, pRb is essential for the maintainance of the postmitotic state of terminally differentiated keratinocytes, preventing cell cycle re-entry. However, p107 compensates for the effects of Rb loss as the phenotypic abnormalities of Rb(F19/F19);K14cre keratinocytes in vivo and in vitro become more severe with the concurrent loss of p107 alleles. p107 alone appears to be dispensable for all these phenotypic changes, as the presence of a single Rb allele in a p107-null background rescues all these alterations. Luciferase reporter experiments indicate that these phenotypic alterations might be mediated by increased E2F activity. Our findings support a model in which pRb in conjunction with p107 plays a central role in regulating epidermal homeostasis.     

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.     

Abnormal epidermal differentiation and impaired epithelial-mesenchymal tissue interactions in mice lacking the retinoblastoma relatives p107 and p130

The functions of p107 and p130, members of the retinoblastoma family, include the control of cell cycle progression and differentiation in several tissues. Our previous studies suggested a role for p107 and p130 in keratinocyte differentiation in vitro. We now extend these data using knockout animal models. We found impaired terminal differentiation in the interfollicular keratinocytes of p107/p130-double-null mice epidermis. In addition, we observed a decreased number of hair follicles and a clear developmental delay in hair, whiskers and tooth germs. Skin grafts of p107/p130-deficient epidermis onto NOD/scid mice showed altered differentiation and hyperproliferation of the interfollicular keratinocytes, thus demonstrating that the absence of p107 and p130 results in the deficient control of differentiation in keratinocytes in a cell-autonomous manner. Besides normal hair formation, follicular cysts, misoriented and dysplastic follicles, together with aberrant hair cycling, were also observed in the p107/p130 skin transplants. Finally, the hair abnormalities in p107/p130-null skin were associated with altered Bmp4-dependent signaling including decreased DeltaNp63 expression. These results indicate an essential role for p107 and p130 in the epithelial-mesenchimal interactions.     

The Rb2/p130 gene product is a nuclear protein whose phosphorylation is cycle regulated

The Rb2/p130 protein has been shown to have a high sequence homology with the retinoblastoma gene product (pRb), one of the most well-characterized tumor suppressor genes, and with pRb-related p107, especially in their conserved pocket domains, which display a primary role in the function of these proteins. In this study, we report on the biochemical and immunocytochemical characterization of the Rb2/p130 protein, using a polyclonal antibody developed against its “spacer” region included in the pocket domain of the whole protein. We show that pRb/p130 is a phosphoprotein located at the nuclear level and that its phosphorylation pathway can be dramatically reduced by phosphatase treatment. Moreover pRb/p130, with p107, with p107, is one of the major targets of the E1A viral oncoprotein-associated kinase activity, showing a phosphorylation pattern which is modulated during the cell cycle, reaching a peak of activation at the onset of S-phase. © 1995 Wiley-Liss, Inc.     

Interaction Between the Cdk2/Cyclin A Complex and a Small Molecule Derived from the pRb2/p130 Spacer Domain: A Theoretical Model

Retinoblastoma (RB) family proteins pRb, p107 and pRb2/p130 are important cellular factors which play a well-recognized role as tumor and growth suppressors. These proteins are actively involved in the negative control of the cell cycle and their function is modulated via complex homeostatic processes, most of them involving post-translational regulation of their phosphorylation status. Interestingly, the family members p107 and pRb2/p130 share the ability to physically interact and inhibit the kinase activity of the Cdk2/Cyclin A and Cdk2/Cyclin E complexes. Regarding pRb2/p130, its inhibitory effect on the Cdk2/Cyclin A activity has been attributed to the “spacer” region. Recently, a 39 aa-long pRb2/p130 spacer-derived peptide (Spa310, aa 641-679) has been selected as the sequence responsible for Cdk2/Cyclin A inhibition. Following the identification of this active sequence, here we propose a computer-generated three-dimensional model of the interaction between the Cdk2/Cyclin A complex and the N-terminal 9-amino acid sequence of the Spa310 peptide. We believe this model as useful for the rational development of peptide or peptidomimetic kinase inhibitors to be used for the negative modulation of cell cycle in cancer cells.     

Transforming growth factor beta inhibits the phosphorylation of pRB at multiple serine/threonine sites and differentially regulates the formation of pRB family-E2F complexes in human myeloid leukemia cells

Transforming growth factor beta (TGFbeta)1 induced dephosphorylation of pRb at multiple serine and threonine residues including Ser249/Thr252, Thr373, Ser780, and Ser807/811 in MV4-11 cells. Likewise, TGFbeta1 caused the dephosphorylation of p130, while inhibiting accumulation of p107 protein. Phosphorylated pRb was detected to bind E2F-1 and E2F-3, which appears to be a major form of pRb complexes in actively cycling cells. TGFbeta1 significantly downregulated pRb-E2F-1 and pRb-E2F-3 complexes as a result of inhibition of E2F-1 and E2F-3. In contrast, complexes of E2F-4 with pRb and with p130 were increased markedly upon TGFbeta1 treatment, whereas p107 associated E2F-4 was dramatically decreased. In agreement with these results, p130-E2F-4 DNA binding activity was dominant in TGFbeta1 treated cells, whereas p107-E2F-4 DNA binding activity was only found in proliferating cells. Our data strongly suggest that inhibition of E2Fs and differential regulation of pRb family-E2F-4 complexes are linked to TGFbeta1-induced growth inhibition. E2F-4 is switched from p107 to p130 and pRb when cells are arrested in G1 phase by TGFbeta1.     

The RB-related gene Rb2/p130 in neuroblastoma differentiation and in B-myb promoter down-regulation

The retinoblastoma family of nuclear factors is composed of RB, the prototype of the tumour suppressor genes and of the strictly related genes p107 and Rb2/p130. The three genes code for proteins, namely pRb, p107 and pRb2/p130, that share similar structures and functions. These proteins are expressed, often simultaneously, in many cell types and are involved in the regulation of proliferation and differentiation. We determined the expression and the phosphorylation of the RB family gene products during the DMSO-induced differentiation of the N1E-115 murine neuroblastoma cells. In this system, pRb2/p130 was strongly up-regulated during mid-late differentiation stages, while, on the contrary, pRb and p107 resulted markedly decreased at late stages. Differentiating N1E-115 cells also showed a progressive decrease in B-myb levels, a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection of each of the RB family genes in these cells was able, at different degrees, to induce neuronal differentiation, to inhibit [3H]thymidine incorporation and to down-regulate the activity of the B-myb promoter.     

Tumor suppressor pRb2/p130 gene and its derived product Spa310 spacer domain as perspective candidates for cancer therapy

Tumor suppressor pRb2/p130 gene belongs to the retinoblastoma (Rb) gene family, which also includes pRb/p105 and pRb/p107. The members of the Rb gene family have attracted a great deal of interest because of their essential role in regulating cell cycle and, consequently, cell proliferation. This mini review discusses the potential therapeutic applications both of pRb2/p130 and its derived product Spa310 spacer domain in cancer treatment.