Inhibition of pluripotency networks by the Rb tumor suppressor restricts reprogramming and tumorigenesis.

Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function.

The extreme COOH terminus of the retinoblastoma tumor suppressor protein pRb is required for phosphorylation on Thr-373 and activation of E2F.

The retinoblastoma protein pRb plays a pivotal role in G(1)- to S-phase cell cycle progression and is among the most frequently mutated gene products in human cancer. Although much focus has been placed on understanding how the A/B pocket and COOH-terminal domain of pRb cooperate to relieve transcriptional repression of E2F-responsive genes, comparatively little emphasis has been placed on the function of the NH(2)-terminal region of pRb and the interaction of the multiple domains of pRb in the full-length context. Using "reverse mutational analysis" of Rb(DeltaCDK) (a dominantly active repressive allele of Rb), we have previously shown that restoration of Thr-373 is sufficient to render Rb(DeltaCDK) sensitive to inactivation via cyclin-CDK phosphorylation. This suggests that the NH(2)-terminal region plays a more critical role in pRb regulation than previously thought. In the present study, we have expanded this analysis to include additional residues in the NH(2)-terminal region of pRb and further establish that the mechanism of pRb inactivation by Thr-373 phosphorylation is through the dissociation of E2F. Most surprisingly, we further have found that removal of the COOH-terminal domain of either RbDeltaCDK(+T373) or wild-type pRb yields a functional allele that cannot be inactivated by phosphorylation and is repressive of E2F activation and S-phase entry. Our data demonstrate a novel function for the NH(2)-terminal domain of pRb and the necessity for cooperation of multiple domains for proper pRb regulation.

Reverse mutational analysis reveals threonine-373 as a potentially sufficient phosphorylation site for inactivation of the retinoblastoma tumor suppressor protein (pRB).

Previous studies in our laboratory have shown that constitutive cyclin E expression can alleviate the requirement for cyclin D-CDK activity in the inactivation of the retinoblastoma protein (pRb). Rb(DeltaCDK), a mutant construct of pRb with 15 of the 16 CDK phosphorylation sites mutated to alanine represses activation of E2F by mitogen, despite cyclin E overexpression. However, restoration of the four cyclin E-CDK2 phosphorylation sites to Rb(DeltaCDK) renders this construct sensitive to inactivation by CDK phosphorylation. In the present study, we engage a "reverse mutational analysis" by restoring cyclin E-CDK2 phosphorylation sites to Rb(DeltaCDK) individually and in combinations in an attempt to discover phosphorylation sites on Rb that are critical for inactivation. Surprisingly, we report that, in both rodent and human cells, restoration of threonine-373 to Rb(DeltaCDK), alone or in combination with other phospho-restorations, results in a loss of the constitutively repressive effect of this construct on E2F activation. Further, induction of endogenous cyclin A protein is blocked by Rb(DeltaCDK), but not by mutants of Rb(DeltaCDK) containing a restored threonine-373. Finally, while S phase entry is blocked by expression of Rb(DeltaCDK), restoration of threonine-373 largely attenuates this effect. These findings reveal that phosphorylation of threonine-373 by CDK2-cyclin E represent a potentially crucial event in the inactivation of the pRb protein.

Retinoblastoma suppression of matrix metalloproteinase 1, but not interleukin-6, through a p38-dependent pathway in rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by increased synovial lining cellularity, inflammation, and destruction of cartilage and bone. During the pathogenesis of RA, synovial fibroblasts reenter the cell cycle and multiply in number. RA synovial fibroblasts express high levels of the MAP kinase p38, which may contribute to the production of interleukin-6 (IL-6) and matrix metalloproteinases (MMPs). IL-6 and MMP-1 promote inflammation and joint destruction, respectively. Taken together, these findings indicate that in RA the enhanced cell cycle activity and production of IL-6 and MMP-1 may be linked. Therefore, we sought to determine if the tumor suppressor gene product retinoblastoma (Rb), a negative regulator of cell cycle activity, inhibits IL-6, MMP-1, and p38 in RA synovial fibroblasts. METHODS: RA and non-RA synovial fibroblasts were examined by enzyme-linked immunosorbent assay (ELISA) for the relative expression of inactive hyperphosphorylated Rb (inactive Rb/total Rb). Ectopic Rb expression was mediated by infection with a replication-defective adenovirus that expresses Rb (Ad-Rb). A control replication-defective adenovirus that expresses beta-galactosidase (Ad-beta-gal) was used. Cell cycle activity was determined by flow cytometry. IL-6 and MMP-1 expression was examined by real-time polymerase chain reaction and ELISA. Expression and activation of p38 were determined by kinase assays and ELISA. The activity of p38 was enhanced by infecting RA synovial fibroblasts with a replication-defective adenovirus that expresses a constitutively active form of MAPK kinase 3 (Ad-CA-MKK3), an upstream activator of p38. RESULTS: Quiescent RA, compared with non-RA synovial fibroblasts, displayed a 200% (P < 0.02) increase in the inactive Rb isoform. Proliferating RA synovial fibroblasts exhibited a 60% (P < 0.12) increase in the inactive Rb isoform compared with non-RA synovial fibroblasts. Increased levels of the active Rb isoform inhibited cell cycle progression and suppressed IL-6 and MMP-1 secretion in RA synovial fibroblasts, although the steady-state levels of IL-6 and MMP-1 messenger RNA remained unchanged. However, Rb overexpression had no effect on spontaneous or IL-1beta-induced production of IL-6 or MMP-1 in non-RA synovial fibroblasts. Ectopic Rb expression reduced the activity of p38. Ad-CA-MKK3 infection in RA synovial fibroblasts increased p38 phosphorylation, and MMP-1 but not IL-6 secretion. In contrast, Rb overexpression inhibited Ad-CA-MKK3-mediated phosphorylation of p38 and subsequent increase in MMP-1. CONCLUSION: Rb-mediated suppression of IL-6 and MMP-1 occurs at a posttranscriptional level. However, Ad-Rb reduction of MMP-1 but not IL-6 requires inhibition of the p38 pathway. These results suggest that Rb negatively regulates p38 activation, leading to decreased MMP-1 secretion in RA synovial fibroblasts.