Biomedical subjects

Lisa J Robinson

Publications and source records attributed to Lisa J Robinson.

5 recordsLinked to original sources

PubMed2005

Osteoclast signalling pathways.

The osteoclast is a monocyte-derived cell with complex regulatory control due to its role, balancing calcium homeostasis with skeletal modelling and repair. Normal differentiation requires tyrosine kinase- and tumor necrosis-family receptors, normally fms and RANK. Ligands for these receptors plus unidentified serum or cell-presented factor(s) are needed for in vitro differentiation, possibly signalling via an immune-like tyrosine kinase acceptor molecule. Osteoclast development and activity are increased by cytokines signalling through GP130, such as IL-6, by TGF-beta, and by IL-1, although these cannot replace serum. Other tyrosine kinase receptors including kit and met can augment fms signalling, and TNFs other than RANKL, including TNFalpha and TRAIL, modify RANK signalling, which is also susceptible to interference by interferons. The situation is further complicated by G-protein coupled receptors including the calcitonin receptor, by integrin or calcium-mediated signals, and by estrogen receptors, which operate in bone largely via NO downstream signals. Differentiation, activity, and survival signals merge in intracellular second messengers. These include cytoplasmic kinases of several families; differentiation pathways often terminate in Erk/Jun kinases or NF-kappaB. Key regulatory intermediates include TRAF6, src, Smad3, phosphatidylinositol-3-kinase, Jak/Stat, and the cGMP-dependent protein kinase I. There are substantial uncertainties regarding how intracellular agents connect to primary signals. The frontier includes characterization of how scaffolding/adapter proteins, such as cbl, gab, grb, p130Cas, and shc, as well as itam-containing proteins and nonreceptor tyrosine kinase adapters of the src and syk families, delimit and integrate signals of multiple receptors to bring about specific outcomes.

Animals↗

PubMed2005

Negative regulation of RANKL-induced osteoclastic differentiation in RAW264.7 Cells by estrogen and phytoestrogens.

We studied estrogen effects on osteoclastic differentiation using RAW264.7, a murine monocytic cell line. Differentiation, in response to RANKL and colony-stimulating factor 1, was evaluated while varying estrogen receptor (ER) stimulation by estradiol or nonsteroidal ER agonists was performed. The RAW264.7 cells were found to express ERalpha but not ERbeta. In contrast to RANKL, which decreased ERalpha expression and induced osteoclast differentiation, 10 nm estradiol, 3 microm genistein, or 3 microm daidzein all increased ERalpha expression, stimulated cell proliferation, and decreased multinucleation, with the effects of estrogen > or = daidzein > genistein. However, no estrogen agonist reduced RANKL stimulation of osteoclast differentiation markers or its down-regulation of ERalpha expression by more than approximately 50%. Genistein is also an Src kinase antagonist in vitro, but it did not decrease Src phosphorylation in RAW264.7 cells relative to other estrogen agonists. However, both phytoestrogens and estrogen inhibited RANKL-induced IkappaB degradation and NF-kappaB nuclear localization with the same relative potency as seen in proliferation and differentiation assays. This study demonstrates, for the first time, the direct effects of estrogen on osteoclast precursor differentiation and shows that, in addition to effecting osteoblasts, estrogen may protect bone by reducing osteoclast production. Genistein, which activates ERs selectively, inhibited osteoclastogenesis less effectively than the nonselective phytoestrogen daidzein, which effectively reproduced effects of estrogen.

Animals↗

PubMed2005

Src family tyrosine kinases are activated by Flt3 and are involved in the proliferative effects of leukemia-associated Flt3 mutations.

OBJECTIVE: The hematopoietic growth factor receptor, Fms-like tyrosine kinase-3 (Flt3), modulates survival and proliferation of myeloid and B-cell precursors. Activating mutations of Flt3 are the most common molecular abnormalities in acute myeloid leukemia (AML) and have an apparent role in leukemogenesis. However, signaling pathways mediating Flt3 effects are incompletely understood. The role of Src kinases is unknown, although some, such as Lyn, have also been linked to leukemogenesis. This study examines the role of Src kinases in Flt3 signaling and the oncogenic effects of leukemia-associated Flt3 mutations. MATERIALS AND METHODS: We examined the activation and functional roles of Src kinases in human leukemic myeloid cell lines expressing wild-type Flt3 or a constitutively active mutant, and in cells stably transduced with human wild-type or mutant Flt3. RESULTS: Flt3 ligand stimulation of wild-type Flt3 increased phosphorylation of Src kinase Lyn. Constitutive Lyn phosphorylation and activation was found in cells expressing constitutively active Flt3 mutants. Src kinases are implicated in downregulation of closely related receptors, but Src inhibitors had no effect on ligand-stimulated Flt3 degradation, or on the rapid degradation of an Flt3 mutant. However, growth-factor-independent proliferation resulting from mutant Flt3 expression did depend on the activity of Src kinases. CONCLUSION: Our studies reveal for the first time the involvement of Src kinases in Flt3 signaling, with activation of Lyn by constitutively active Flt3 mutants as well as ligand-stimulated wild-type receptor, and show that Src kinase inhibitors block proliferative effects of Flt3 mutants found in AML. Thus, Src kinases may represent targets for inhibitor therapy in Flt3-related AML.

Cell Line, Tumor↗

PubMed2005

Complete remission following clofarabine treatment in refractory juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is the most common myeloproliferative/myelodysplastic disorder seen in children. The treatment of choice, allogeneic stem cell transplantation, provides the only known cure for the disease, but relapse after transplant is common. The authors describe a 5-year-old boy diagnosed at age 34 months with JMML that evolved to acute myeloid leukemia. Initial treatment consisted of fludarabine and cis-retinoic acid therapy, followed by a matched sibling bone marrow transplant. After a relapse, he received a second transplant from the same donor, using peripheral blood stem cells, followed by repeated donor leukocyte infusions. After the second relapse, he received the farnesyltransferase inhibitor R115777 (tipifarnib, Zarnestra), but the leukemia persisted. When bone marrow blasts numbered 60% of the mononuclear cells, he received single-agent clofarabine induction (52 mg/m/d) for 5 days. After three courses, he attained a remission marrow with 5% blasts and disappearance of the 5q- and 9q- cytogenetic abnormalities.

Adenine Nucleotides↗

PubMed2003

G-CSF-induced tyrosine phosphorylation of Gab2 is Lyn kinase dependent and associated with enhanced Akt and differentiative, not proliferative, responses.

The granulocyte colony-stimulating factor receptor (G-CSFR) transduces intracellular signals for myeloid cell proliferation, survival, and differentiation through the recruitment of nonreceptor protein tyrosine kinases Lyn and janus kinase 2 (Jak2). This results in the tyrosine phosphorylation of a small set of positive and negative adapters and effectors. Grb2-associated binder-2 (Gab2) is a newly described adapter molecule, preferentially expressed in hematopoietic cells and associated with phosphatidylinositol 3 (PI3) kinase. Studies suggest that Gab2 plays both positive and negative roles in cytokine receptor signaling. To investigate the role Gab2 plays in G-CSF receptor-mediated signaling, we have analyzed its activation state and correlated that with wild-type and mutant G-CSF receptors stably expressed in the murine factor-dependent Ba/F3 cell lines. G-CSF-induced tyrosine phosphorylation of Gab2 occurred in the wild-type and single Y-to-F mutants (Y704F, Y729F, and Y744F), but not in the ADA and W650R loss-of-function mutants. Cells expressing truncated proximal G-CSFR, the tyrosine-null (Y4F) G-CSFR, or Y764F mutant receptors had decreased phosphorylation of Gab2. Specific inhibitors of Src kinase (PD173 and PP1) but not Jak2 kinase (AG490) blocked Gab2 phosphorylation. Phosphorylation of Gab2 occurred in wild-type, but not Lyn-deficient, G-CSFR-transfected DT40 B cells. These data propose that Lyn, not Jak2, phosphorylates Gab2 and that maximal phosphorylation of Gab2 requires Y764, a Grb2-binding site. Serine phosphorylation of Akt, a marker of PI3-kinase activity, was detected in both wild-type and truncated proximal domain receptors, but not in the ADA and W650R mutants. Levels of phospho-Akt and phospho-extracellular signal-regulated kinase (phospho-ERK) were greater in proximal truncated than in wild-type G-CSFR cells, suggesting that Gab2 is dissociated from PI3 kinase or ERK activities. Overexpression of Gab2 enhanced the phosphorylation state of Akt, but not of ERK. This inhibited the proliferation of wild-type and truncated G-CSFR-transfected Ba/F3 cells and enhanced their myeloid differentiation. All together, these data indicate that G-CSF treatment leads to Lyn-mediated tyrosine phosphorylation of Gab2, which may serve as an important intermediate of enhanced Akt activity and myeloid differentiation, not growth/survival response.

Adaptor Proteins, Signal Transducing↗