Lucie Canaff

PTH-related peptide (PTHrP) and its receptor, the PTH/PTHrP receptor type 1 (PTH1R), are essential for chondrocyte differentiation and epiphyseal cartilage homeostasis. To evaluate the functional differences between PTHrP and PTH1R derived from mesenchymal progenitor cells (MPCs) in skeletal development, we generated MPC-specific Pthrp conditional KO mice (PTHrP cKO; Prrx1-Cre; Pthrpflox/flox) or Pth1r cKO mice (PTH1R cKO; Prrx1-Cre; Pth1rflox/flox) using the Prrx1-Cre driver and analyzed their skeletal phenotypes. Both PTHrP cKO mice and PTH1R cKO mice exhibited severe shortening of long bones and impaired growth plate formation immediately after birth. However, in the digits, only PTHrP cKO mice showed distinctive digit deviations in the toes, which were similar to those seen in human diastrophic dysplasia (DTD). These results suggest that MPC-derived PTHrP and PTH1R play distinct roles. Both are essential for longitudinal bone growth, but PTHrP plays an important role in regulating toe position and morphology. This phenotypic difference suggests a novel PTH1R-independent function of PTHrP in skeletal development. Thus, our study provides genetic evidence for this mechanism, offering insights into the pathogenesis of congenital digit deformities such as those seen in DTD.

Over the past few decades, the worldwide incidence of cutaneous melanoma, a malignant neoplasm arising from melanocytes, has been increasing markedly, leading to the highest rate of skin cancer-related deaths. While localized tumors are easily removed by excision surgery, late-stage metastatic melanomas are refractory to treatment and exhibit a poor prognosis. Consequently, unraveling the molecular mechanisms underlying melanoma tumorigenesis and metastasis is crucial for developing novel targeted therapies. We found that the multiple endocrine neoplasia type 1 (MEN1) gene product Menin is required for the transforming growth factor beta (TGFβ) signaling pathway to induce cell growth arrest and apoptosis in vitro and prevent tumorigenesis in vivo in preclinical xenograft models of melanoma. We further identified point mutations in two MEN1 family members affected by melanoma that led to proteasomal degradation of the MEN1 gene product and to a loss of TGFβ signaling. Interestingly, blocking the proteasome degradation pathway using an FDA-approved drug and RNAi targeting could efficiently restore MEN1 expression and TGFβ transcriptional responses. Together, these results provide new potential therapeutic strategies and patient stratification for the treatment of cutaneous melanoma.

Bone has a complex microenvironment formed by an extracellular matrix (ECM) composed mainly of mineralized type I collagen fibres. Bone ECM regulates signaling pathways important in the differentiation of osteoblast-lineage cells, necessary for bone mineralization and in preserving tissue architecture. Compared to conventional 2D cell cultures, 3D in vitro models may better mimic bone ECM and provide an environment to support osteoblastic differentiation. In this study, a biomimetic 3D osteoid-like dense collagen gel model was used to investigate the role of the nuclear protein menin plays in osteoblastic differentiation and matrix mineralization. Previous in vitro and in vivo studies have shown that when expressed at later stages of osteoblastic differentiation, menin modulates osteoblastogenesis and regulates bone mass in adult mice. To investigate the role of menin when expressed at earlier stages of the osteoblastic lineage, conditional knockout mice in which the Men1 gene is specifically deleted early (i.e., at the level of the pluripotent mesenchymal stem cell lineage), where generated and primary calvarial osteoblasts were cultured in plastically compressed dense collagen gels for 21 days. The proliferation, morphology and differentiation of isolated seeded primary calvarial osteoblasts from knockout (Prx1-Cre; Men1f/f) mice were compared to those isolated from wild-type (Men1f/f) mice. Primary calvarial osteoblasts from knockout and wild-type mice did not show differences in terms of proliferation. However, in comparison to wild-type cells, primary osteoblast cells derived from knockout mice demonstrated deficient mineralization capabilities and an altered gene expression profile when cultured in 3D dense collagen gels. In summary, these findings indicate that when expressed at earlier stages of osteoblast differentiation, menin is important in maintaining matrix mineralization in 3D dense collagen gel matrices, in vitro.

Menin, the product of the Men1 tumor suppressor gene, facilitates the cell proliferation control and differentiation induced by the TGF-beta superfamily of ligands critical for bone development and maintenance. Our in vivo studies have shown the importance of menin for proper functioning of the mature osteoblast and maintenance of bone mass in adult mice. In the present study, we examined the in vivo role of menin at earlier stages of the osteoblast lineage through conditional knockout of the Men1 gene. This was implemented through the Cre-LoxP recombination system at the level of the osteochondroprogenitor, osteoblast progenitor as well as, for comparison, the mature osteoblast. Prx1-Cre;Men1f/f, Osx-Cre;Men1f/f and OC-Cre;Men1f/f mice represent knockout of the Men1 gene in the mesenchymal stem cell, the preosteoblast and the mature osteoblast, respectively. Mice were sacrificed and analyzed at six months of age. All experiments had institutional ethical approval. Prx1-Cre;Men1f/f and Osx-Cre;Men1f/f mice were smaller than wild-type littermates whereas OC-Cre;Men1f/f mice were of normal size. Femur lengths of Prx1-Cre;Men1f/f and Osx-Cre;Men1f/f mice were shorter whereas those of OC-Cre;Men1f/f mice were of normal length. Prx1-Cre;Men1f/f and Osx-Cre;Men1f/f mice had reduced BMD (dual energy X-ray absorptiometry) whereas that of OC-Cre;Men1f/f mice was normal. By 3-dimensional micro-CT imaging of femur, all three strains of Men1 knockout mice had decreased trabecular bone volume with altered trabecular structure and decreased cortical bone thickness. In all strains of knockout mice trabecular number and spacing were decreased and increased, respectively. Primary calvarial osteoblasts of all strains of knockout mice relative to those of wild-type mice were deficient in mineralization and differentiation as assessed by Alizarin red, von Kossa and alkaline phosphatase staining, and had impaired responsiveness to TGF-beta and BMP-2 in specific promoter-reporter transfection assays. In conclusion, menin plays a crucial role in the development as well as maintenance of bone mass.

Cutaneous melanoma is the most lethal skin cancer and its incidence in developed countries has dramatically increased over the past decades. Localized tumors are easily treated by surgery, but advanced melanomas lack efficient treatment and are associated with very poor outcomes. Thus, understanding the processes underlying melanoma development and progression is critical. The Transforming Growth Factor beta (TGFβ) acts as a potent tumor suppressor in human melanoma, by inhibiting cell growth and preventing cellular migration and invasion. In this study, we aimed at elucidating the molecular mechanisms underlying TGFβ-mediated tumor suppression. Human cutaneous melanoma cell lines, derived from different patients, were used to assess for cell cycle analysis, apoptosis/caspase activity and cell migration. Techniques involved immunoblotting, immunohistochemistry, real time PCR and luciferase reporter assays. We found the leukemia inhibitory factor (LIF) to be strongly up-regulated by TGFβ in melanoma cells, defining LIF as a novel TGFβ downstream target gene in cutaneous melanoma. Interestingly, we also showed that TGFβ-mediated LIF expression is required for TGFβ-induced cell cycle arrest and caspase-mediated apoptosis, as well as for TGFβ-mediated inhibition of cell migration. Moreover, we found that TGFβ-mediated LIF expression leads to activation of transcription of the cell cycle inhibitor p21 in a STAT3-dependent manner, and further showed that p21 is required for TGFβ/LIF-mediated cell cycle arrest and TGFβ-induced gene activation of several pro-apoptotic genes. Together, our results define the LIF/p21 signaling cascade as a novel tumor suppressive-like pathway in melanoma, acting downstream of TGFβ to regulate cell cycle arrest and cell death, further highlight new potential therapeutic strategies for the treatment of cutaneous melanoma.

Menin function is related to transcriptional regulation and cell cycle control and it physicallyand functionally interacts with osteotropic transcription factors, such as Smad1/5, Smad3, Runx2 and JunD. Menin promotes the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage, mediated by interactions between menin and the BMP signaling molecules, Smad1/S, or Runx2. On the other hand, in mature osteoblasts the interaction of menin and the TGF-β/Smad3 pathway counteracts the BMP-2/Smad1/S- and Runx2-induced transcriptional activities leading to inhibition of late stage osteoblast differentiation. Moreover, menin suppresses osteoblast maturation partly by inhibiting the differentiation actions of JunD. In conclusion, menin plays an important role in osteoblastogenesis and osteoblast differentiation. Keywords Mesenchymal Stem Cell Transform Growth Factor Osteoblast Differentiation Multiple Endocrine Neoplasia Type Osteoblast Lineage These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.