Joy Y. Wu

Osteoblastic regulation of B lymphopoiesis is mediated by Gsα-dependent signaling pathways

Osteoblasts play an increasingly recognized role in supporting hematopoietic development and recently have been implicated in the regulation of B lymphopoiesis. Here we demonstrate that the heterotrimeric G protein α subunit Gsα is required in cells of the osteoblast lineage for normal postnatal B lymphocyte production. Deletion of Gsα early in the osteoblast lineage results in a 59% decrease in the percentage of B cell precursors in the bone marrow. Analysis of peripheral blood from mutant mice revealed a 67% decrease in the number of circulating B lymphocytes by 10 days of age. Strikingly, other mature hematopoietic lineages are not decreased significantly. Mice lacking Gsα in cells of the osteoblast lineage exhibit a reduction in pro-B and pre-B cells. Furthermore, interleukin (IL)-7 expression is attenuated in Gsα-deficient osteoblasts, and exogenous IL-7 is able to restore B cell precursor populations in the bone marrow of mutant mice. Finally, the defect in B lymphopoiesis can be rescued by transplantation into a WT microenvironment. These findings confirm that osteoblasts are an important component of the B lymphocyte niche and demonstrate in vivo that Gsα-dependent signaling pathways in cells of the osteoblast lineage extrinsically regulate bone marrow B lymphopoiesis, at least partially in an IL-7-dependent manner.

Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4.

Ca2+/calmodulin-dependent protein kinase IV (Camk4; also known as CaMKIV), a multifunctional serine/threonine protein kinase with limited tissue distribution, has been implicated in transcriptional regulation in lymphocytes, neurons and male germ cells. In the mouse testis, however, Camk4 is expressed in spermatids and associated with chromatin and nuclear matrix. Elongating spermatids are not transcriptionally active, raising the possibility that Camk4 has a novel function in male germ cells. To investigate the role of Camk4 in spermatogenesis, we have generated mice with a targeted deletion of the gene Camk4. Male Camk4−/− mice are infertile with impairment of spermiogenesis in late elongating spermatids. The sequential deposition of sperm basic nuclear proteins on chromatin is disrupted, with a specific loss of protamine-2 and prolonged retention of transition protein-2 (Tnp2) in step-15 spermatids. Protamine-2 is phosphorylated by Camk4 in vitro, implicating a connection between Camk4 signalling and the exchange of basic nuclear proteins in mammalian male germ cells. Defects in protamine-2 have been identified in sperm of infertile men, suggesting that our results may have clinical implications for the understanding of human male infertility.

Differential regulation of myeloid leukemias by the bone marrow microenvironment

Like their normal hematopoietic stem cell counterparts, leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) are presumed to reside in specific niches in the bone marrow microenvironment (BMM) and may be the cause of relapse following chemotherapy. Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. CD44 (refs. 3,4) and interleukin-6 (ref. 5) have been implicated previously in the LSC niche. Transforming growth factor-β1 (TGF-β1) is released during bone remodeling and plays a part in maintenance of CML LSCs, but a role for TGF-β1 from the BMM has not been defined. Here, we show that alteration of the BMM by osteoblastic cell–specific activation of the parathyroid hormone (PTH) receptor attenuates BCR-ABL1 oncogene–induced CML-like myeloproliferative neoplasia (MPN) but enhances MLL-AF9 oncogene–induced AML in mouse transplantation models, possibly through opposing effects of increased TGF-β1 on the respective LSCs. PTH treatment caused a 15-fold decrease in LSCs in wild-type mice with CML-like MPN and reduced engraftment of immune-deficient mice with primary human CML cells. These results demonstrate that LSC niches in CML and AML are distinct and suggest that modulation of the BMM by PTH may be a feasible strategy to reduce LSCs, a prerequisite for the cure of CML.

Myelopoiesis is regulated by osteocytes through Gsα-dependent signaling

Hematopoietic progenitors are regulated in their respective niches by cells of the bone marrow microenvironment. The bone marrow microenvironment is composed of a variety of cell types, and the relative contribution of each of these cells for hematopoietic lineage maintenance has remained largely unclear. Osteocytes, the most abundant yet least understood cells in bone, are thought to initiate adaptive bone remodeling responses via osteoblasts and osteoclasts. Here we report that these cells regulate hematopoiesis, constraining myelopoiesis through a Gsα-mediated mechanism that affects G-CSF production. Mice lacking Gsα in osteocytes showed a dramatic increase in myeloid cells in bone marrow, spleen, and peripheral blood. This hematopoietic phenomenon was neither intrinsic to the hematopoietic cells nor dependent on osteoblasts but was a consequence of an altered bone marrow microenvironment imposed by Gsα deficiency in osteocytes. Conditioned media from osteocyte-enriched bone explants significantly increased myeloid colony formation in vitro, which was blocked by G-CSF–neutralizing antibody, indicating a critical role of osteocyte-derived G-CSF in the myeloid expansion.

Role of the Osteoblast Lineage in the Bone Marrow Hematopoietic Niches

In mammals, hematopoiesis shifts to the bone marrow in late embryogenesis, coincident with the appearance of a marrow cavity.(1) There hematopoiesis is sustained throughout adult life unless forced out of the bone marrow into extramedullary sites by pathological conditions. All hematopoietic lineages arise from the hematopoietic stem cell (HSC), and maintenance of HSC self-renewal and differentiation are critically dependent on the presence of a supportive microenvironment, or niche. The existence of such a niche within the bone marrow was first postulated by Schofield,(2) and the vital role of the bone marrow microenvironment has been convincingly shown in the three decades since. The marrow microenvironment is comprised of cells of multiple lineages, including fibroblast-like cells, adipocytes, osteoblasts, and endothelial cells. Recent studies have begun to highlight the contributions of individual lineages to the hematopoietic niche, with the greatest weight of evidence thus far in support of important roles for osteoblasts and the vasculature.(3–5) In humans, CD146+ subendothelial cells have been reported to serve as skeletal progenitors capable of generating cells that organize a hematopoietic microenvironment on transplantation.(6) Subsequent studies have shown that the endosteal surface is rich in vasculature with close approximation of osteoblasts and vessel walls.(7,8) In trabecular bone, it is unlikely that there are physically distinct endosteal and perivascular/vascular niches, although the functional roles of osteoblasts and vascular cells may well differ. This review will focus specifically on the role of cells of the osteoblast lineage within the bone marrow niche. In particular, we will examine the contribution of osteoblasts in supporting hematopoietic stem cells and contrast this with how distinct stages of osteoblast precursors support developing B lymphocytes, one of the best-characterized specific hematopoietic lineages.

Gsα enhances commitment of mesenchymal progenitors to the osteoblast lineage but restrains osteoblast differentiation in mice

The heterotrimeric G protein subunit Gsα stimulates cAMP-dependent signaling downstream of G protein-coupled receptors. In this study, we set out to determine the role of Gsα signaling in cells of the early osteoblast lineage in vivo by conditionally deleting Gsα from osterix-expressing cells. This led to severe osteoporosis with fractures at birth, a phenotype that was found to be the consequence of impaired bone formation rather than increased resorption. Osteoblast number was markedly decreased and osteogenic differentiation was accelerated, resulting in the formation of woven bone. Rapid differentiation of mature osteoblasts into matrix-embedded osteocytes likely contributed to depletion of the osteoblast pool. In addition, the number of committed osteoblast progenitors was diminished in both bone marrow stromal cells (BMSCs) and calvarial cells of mutant mice. In the absence of Gsα, expression of sclerostin and dickkopf1 (Dkk1), inhibitors of canonical Wnt signaling, was markedly increased; this was accompanied by reduced Wnt signaling in the osteoblast lineage. In summary, we have shown that Gsα regulates bone formation by at least two distinct mechanisms: facilitating the commitment of mesenchymal progenitors to the osteoblast lineage in association with enhanced Wnt signaling; and restraining the differentiation of committed osteoblasts to enable production of bone of optimal mass, quality, and strength.

Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow

Osteocalcin (Ocn)-expressing bone marrow cells produce the Notch ligand DLL4, and this is required for lymphoid progenitor cells to seed the thymus.

Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the bone marrow

Breast cancer cells frequently home to the bone marrow, where they may enter a dormant state before forming a bone metastasis. Several members of the interleukin-6 (IL-6) cytokine family are implicated in breast cancer bone colonization, but the role for the IL-6 cytokine leukaemia inhibitory factor (LIF) in this process is unknown. We tested the hypothesis that LIF provides a pro-dormancy signal to breast cancer cells in the bone. In breast cancer patients, LIF receptor (LIFR) levels are lower with bone metastases and are significantly and inversely correlated with patient outcome and hypoxia gene activity. Hypoxia also reduces the LIFR:STAT3:SOCS3 signalling pathway in breast cancer cells. Loss of the LIFR or STAT3 enables otherwise dormant breast cancer cells to downregulate dormancy-, quiescence- and cancer stem cell-associated genes, and to proliferate in and specifically colonize the bone, suggesting that LIFR:STAT3 signalling confers a dormancy phenotype in breast cancer cells disseminated to bone.

Potent constitutive cyclic AMP-generating activity of XLαs implicates this imprinted GNAS product in the pathogenesis of McCune–Albright Syndrome and fibrous dysplasia of bone

Patients with McCune-Albright syndrome (MAS), characterized primarily by hyperpigmented skin lesions, precocious puberty, and fibrous dyslasia of bone, carry postzygotic heterozygous mutations of GNAS causing constitutive cAMP signaling. GNAS encodes the α-subunit of the stimulatory G protein (Gsα), as well as a large variant (XLαs) derived from the paternal allele. The mutations causing MAS affect both GNAS products, but whether XLαs, like Gsα, can be involved in the pathogenesis remains unknown. Here, we investigated biopsy samples from four previously reported and eight new patients with MAS. Activating mutations of GNAS (Arg201 with respect to the amino acid sequence of Gsα) were present in all the previously reported and five of the new cases. The mutation was detected within the paternally expressed XLαs transcript in five and the maternally expressed NESP55 transcript in four cases. Tissues carrying paternal mutations appeared to have higher XLαs mRNA levels than maternal mutations. The human XLαs mutant analogous to Gsα-R201H (XLαs-R543H) showed markedly higher basal cAMP accumulation than wild-type XLαs in transfected cells. Wild-type XLαs demonstrated higher basal and isoproterenol-induced cAMP signaling than Gsα and co-purified with Gβ1γ2 in transduced cells. XLαs mRNA was measurable in mouse calvarial cells, with its level being significantly higher in undifferentiated cells than those expressing preosteoblastic markers osterix and alkaline phosphatase. XLαs mRNA was also expressed in murine bone marrow stromal cells and preosteoblastic MC3T3-E1 cells. Our findings are consistent with the possibility that constitutive XLαs activity adds to the molecular pathogenesis of MAS and fibrous dysplasia of bone.

Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation

Significance Despite the importance of Wnt signaling in bone biology, there is a knowledge gap in the identity of the cells that produce the Wnt ligands and the functions of Wnts produced by specific cell types. In our study, we comprehensively characterized the expression patterns of all 19 Wnts in the developing mouse bone by in situ hybridization, and further showed that Osterix-expressing cells can produce Wnts and respond to Wnt signaling. Additionally, we found that Wnts produced by these Osterix-expressing cells regulate their differentiation and proliferation. Through providing a better understanding of how Wnt signaling contributes to bone biology, our findings also have clinical implications for the mechanism of the osteoporotic drug that targets Sclerostin, a Wnt signaling antagonist. Wnt signaling is a critical regulator of bone development, but the identity and role of the Wnt-producing cells are still unclear. We addressed these questions through in situ hybridization, lineage tracing, and genetic experiments. First, we surveyed the expression of all 19 Wnt genes and Wnt target gene Axin2 in the neonatal mouse bone by in situ hybridization, and demonstrated—to our knowledge for the first time—that Osterix-expressing cells coexpress Wnt and Axin2. To track the behavior and cell fate of Axin2-expressing osteolineage cells, we performed lineage tracing and showed that they sustain bone formation over the long term. Finally, to examine the role of Wnts produced by Osterix-expressing cells, we inhibited Wnt secretion in vivo, and observed inappropriate differentiation, impaired proliferation, and diminished Wnt signaling response. Therefore, Osterix-expressing cells produce their own Wnts that in turn induce Wnt signaling response, thereby regulating their proliferation and differentiation.