Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression.

Control of organ size by cell proliferation and survival is a fundamental developmental process, and its deregulation leads to cancer. However, the molecular mechanism underlying organ size control remains elusive in vertebrates. In Drosophila, the Hippo (Hpo) signaling pathway controls organ size by both restricting cell growth and proliferation and promoting cell death. Here we investigated whether mammals also require the Hpo pathway to control organ size and adult tissue homeostasis. We found that Mst1 and Mst2, the two mouse homologs of the Drosophila Hpo, control the sizes of some, but not all organs, in mice, and Mst1 and Mst2 act as tumor suppressors by restricting cell proliferation and survival. We show that Mst1 and Mst2 play redundant roles, and removal of both resulted in early lethality in mouse embryos. Importantly, tumors developed in the liver with a substantial increase of the stem/progenitor cells by 6 months after removing Mst1 and Mst2 postnatally. We show that Mst1 and Mst2 were required in vivo to control Yap phosphorylation and activity. Interestingly, apoptosis induced by TNFalpha was blocked in the Mst1 and Mst2 double-mutant cells both in vivo and in vitro. As TNFalpha is a pleiotropic inflammatory cytokine affecting most organs by regulating cell proliferation and cell death, resistance to TNFalpha-induced cell death may also contribute significantly to tumor formation in the absence of Mst1 and Mst2.

Sox9 inhibits Wnt signaling by promoting beta-catenin phosphorylation in the nucleus.

Chondrocyte fate determination and maintenance requires Sox9, an intrinsic transcription factor, but is inhibited by Wnt/beta-catenin signaling activated by extrinsic Wnt ligands. Here we explored the underlying molecular mechanism by which Sox9 antagonizes the Wnt/beta-catenin signaling in chondrocyte differentiation. We found that Sox9 employed two distinct mechanisms to inhibit Wnt/beta-catenin signaling: the Sox9 N terminus is necessary and sufficient to promote beta-catenin degradation, whereas the C terminus is required to inhibit beta-catenin transcriptional activity without affecting its stability. Sox9 binds to beta-catenin and components of the beta-catenin "destruction complex," glycogen synthase kinase 3 and beta-transducin repeat containing protein, to promote their nuclear localization. Independent of its DNA binding ability, nuclear localization of Sox9 is both necessary and sufficient to enhance beta-catenin phosphorylation and its subsequent degradation. Thus, one mechanism whereby Sox9 regulates chondrogenesis is to promote efficient beta-catenin phosphorylation in the nucleus. This mechanism may be broadly employed by other intrinsic cell fate determining transcription factors to promptly turn off extrinsic inhibitory Wnt signaling mediated by beta-catenin.

Wnt5a inhibits canonical Wnt signaling in hematopoietic stem cells and enhances repopulation.

The mechanisms that regulate hematopoietic stem cell (HSC) fate decisions between proliferation and multilineage differentiation are unclear. Members of the Wnt family of ligands that activate the canonical Wnt signaling pathway, which utilizes beta-catenin to relay the signal, have been demonstrated to regulate HSC function. In this study, we examined the role of noncanonical Wnt signaling in regulating HSC fate. We observed that noncanonical Wnt5a inhibited Wnt3a-mediated canonical Wnt signaling in HSCs and suppressed Wnt3a-mediated alterations in gene expression associated with HSC differentiation, such as increased expression of myc. Wnt5a increased short- and long-term HSC repopulation by maintaining HSCs in a quiescent G(0) state. From these data, we propose that Wnt5a regulates hematopoiesis by the antagonism of the canonical Wnt pathway, resulting in a pool of quiescent HSCs.

Wnt/beta-catenin signaling is sufficient and necessary for synovial joint formation.

A critical step in skeletal morphogenesis is the formation of synovial joints, which define the relative size of discrete skeletal elements and are required for the mobility of vertebrates. We have found that several Wnt genes, including Wnt4, Wnt14, and Wnt16, were expressed in overlapping and complementary patterns in the developing synovial joints, where beta-catenin protein levels and transcription activity were up-regulated. Removal of beta-catenin early in mesenchymal progenitor cells promoted chondrocyte differentiation and blocked the activity of Wnt14 in joint formation. Ectopic expression of an activated form of beta-catenin or Wnt14 in early differentiating chondrocytes induced ectopic joint formation both morphologically and molecularly. In contrast, genetic removal of beta-catenin in chondrocytes led to joint fusion. These results demonstrate that the Wnt/beta-catenin signaling pathway is necessary and sufficient to induce early steps of synovial joint formation. Wnt4, Wnt14, and Wnt16 may play redundant roles in synovial joint induction by signaling through the beta-catenin-mediated canonical Wnt pathway.

Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation.

Wnts are secreted signaling molecules that can transduce their signals through several different pathways. Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems. We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin. This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent. Furthermore, we provide evidence that Wnt-5a also acts in vivo to promote beta-catenin degradation in regulating mammalian limb development and possibly in suppressing tumor formation.

Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation.

Proper longitudinal growth of long bones relies on the regulation of specific spatial patterns of chondrocyte proliferation and differentiation. We have studied the roles of two members of the Wnt family, Wnt5a and Wnt5b in long bone development. We show that Wnt5a is required for longitudinal skeletal outgrowth and that both Wnt5a and Wnt5b regulate the transition between different chondrocyte zones independently of the Indian hedgehog (Ihh)/parathyroid hormone-related peptide (PTHrP) negative feedback loop. We find that important cell cycle regulators such as cyclin D1 and p130, a member of the retinoblastoma family, exhibit complimentary expression patterns that correlate with the distinct proliferation and differentiation states of chondrocyte zones. Furthermore, we show that Wnt5a and Wnt5b appear to coordinate chondrocyte proliferation and differentiation by differentially regulating cyclin D1 and p130 expression, as well as chondrocyte-specific Col2a1 expression. Our data indicate that Wnt5a and Wnt5b control the pace of transitions between different chondrocyte zones.