Ying Hua Cheng

Impact of interactions of cellular components of the bone marrow microenvironment on hematopoietic stem and progenitor cell function

Hematopoietic stem (HSC) and progenitor (HPC) cell fate is governed by intrinsic and extrinsic parameters. We examined the impact of hematopoietic niche elements on HSC and HPC function by analyzing the combined effect of osteoblasts (OBs) and stromal cells (SCs) on Lineage(-)Sca-1(+)CD117(+) (LSK) cells. CFU expansion and marrow repopulating potential of cultured Lineage(-)Sca-1(+)CD117(+) cells were significantly higher in OB compared with SC cultures, thus corroborating the importance of OBs in the competence of the hematopoietic niche. OB-mediated enhancement of HSC and HPC function was reduced in cocultures of OBs and SCs, suggesting that SCs suppressed the OB-mediated hematopoiesis-enhancing activity. Although the suppressive effect of SC was mediated by adipocytes, probably through up-regulation of neuropilin-1, the OB-mediated enhanced hematopoiesis function was elaborated through Notch signaling. Expression of Notch 2, Jagged 1 and 2, Delta 1 and 4, Hes 1 and 5, and Deltex was increased in OB cultures and suppressed in SC and OB/SC cultures. Phenotypic fractionation of OBs did not segregate the hematopoiesis-enhancing activity but demonstrated that this function is common to OBs from different anatomic sites. These data illustrate that OBs promote in vitro maintenance of hematopoietic functions, including repopulating potential by up-regulating Notch-mediated signaling between HSCs and OBs.

Osteoblast lineage cells expressing high levels of Runx2 enhance hematopoietic progenitor cell proliferation and function

Although osteoblasts (OB) play a key role in the hematopoietic stem cell (HSC) niche, little is known as to which specific OB lineage cells are critical for the enhancement of stem and progenitor cell function. Unlike hematopoietic cells, OB cell surface phenotypic definitions are not well developed. Therefore, to determine which OB lineage cells are most important for hematopoietic progenitor cell (HPC) function, we characterized OB differentiation by gene expression and OB function, and determined whether associations existed between OB and HPC properties. OB were harvested from murine calvariae, used immediately (fresh OB) or cultured for 1, 2, or 3 weeks prior to their co‐culture with Lin−Sca1+c‐kit+ (LSK) cells for 1 week. OB gene expression, alkaline phosphatase activity, calcium deposition, hematopoietic cell number fold increase, CFU fold increase, and fold increase of Lin−Sca1+ cells were determined. As expected, HPC properties were enhanced when LSK cells were cultured with OB compared to being cultured alone. Initial alkaline phosphatase and calcium deposition levels were significantly and inversely associated with an increase in the number of LSK progeny. Final calcium deposition levels and OB culture duration were inversely associated with all HPC parameters, while Runx2 levels were positively associated with all HPC properties. Since calcium deposition is associated with OB maturation and high levels of Runx2 are associated with less mature OB lineage cells, these results suggest that less mature OB better promote HPC proliferation and function than do more mature OB. J. Cell. Biochem. 111: 284–294, 2010.

Immature and Mature Megakaryocytes Enhance Osteoblast Proliferation and Inhibit Osteoclast Formation

Recent data suggest that megakaryocytes (MKs) play a role in skeletal homeostasis. In vitro and in vivo data show that MKs stimulate osteoblast (OB) proliferation and inhibit osteoclast (OC) formation, thus favoring net bone deposition. There are several mouse models with dysregulated megakaryopoiesis and resultant high bone mass phenotypes. One such model that our group has extensively studied is GATA‐1 deficient mice. GATA‐1 is a transcription factor required for normal megakaryopoiesis, and mice deficient in GATA‐1 have increases in immature MK number and a striking increase in bone mass. While the increased bone mass could simply be a result of increased MK number, here we take a more in depth look at the MKs of these mice to see if there is a unique factor inherent to GATA‐1 deficient MKs that favors increased bone deposition. We show that increased MK number does correspond with increased OB proliferation and decreased OC formation that stage of maturation does not alter the effect of MKs on bone cell lineages beyond the megakaryoblast stage, and that GATA‐1 deficient MKs survive longer than wild‐type controls. So while increased MK number in GATA‐1 deficient mice likely contributes to the high bone mass phenotype, we propose that the increased longevity of this lineage also plays a role. Since GATA‐1 deficient MKs live longer they are able to exert both more proliferative influence on OBs and more inhibitory influence on OCs. J. Cell. Biochem. 109: 774–781, 2010.

Hierarchical organization of osteoblasts reveals the significant role of CD166 in hematopoietic stem cell maintenance and function.

The role of osteoblasts (OB) in maintaining hematopoietic stem cells (HSC) in their niche is well elucidated, but the exact definition, both phenotypically and hierarchically of OB responsible for these functions is not clearly known. We previously demonstrated that OB maturational status influences HSC function whereby immature OB with high Runx2 expression promote hematopoietic expansion. Here, we show that Activated Leukocyte Cell Adhesion Molecule (ALCAM) or CD166 expression on OB is directly correlated with Runx2 expression and high hematopoiesis enhancing activity (HEA). Fractionation of OB with lineage markers: Sca1, osteopontin (OPN), CD166, CD44, and CD90 revealed that Lin-Sca1-OPN+CD166+ cells (CD166+) and their subpopulations fractionated with CD44 and CD90 expressed high levels of Runx2 and low levels of osteocalcin (OC) demonstrating the relatively immature status of these cells. Conversely, the majority of the Lin-Sca1-OPN+CD166- cells (CD166-) expressed high OC levels suggesting that CD166- OB are more mature. In vitro hematopoietic potential of LSK cells co-cultured for 7days with fresh OB or OB pre-cultured for 1, 2, or 3 weeks declined precipitously with increasing culture duration concomitant with loss of CD166 expression. Importantly, LSK cells co-cultured with CD166+CD44+CD90+ OB maintained their in vivo repopulating potential through primary and secondary transplantation, suggesting that robust HEA activity is best mediated by immature CD166+ OB with high Runx2 and low OC expression. These studies begin to define the hierarchical organization of osteoblastic cells and provide a more refined definition of OB that can mediate HEA.

Impact of maturational status on the ability of osteoblasts to enhance the hematopoietic function of stem and progenitor cells.

Osteoblasts (OBs) exert a prominent regulatory effect on hematopoietic stem cells (HSCs). We evaluated the difference in hematopoietic expansion and function in response to co‐culture with OBs at various stages of development. Murine calvarial OBs were seeded directly (fresh) or cultured for 1, 2, or 3 weeks prior to seeding with 1000 Lin‐Sca1 + cKit+ (LSK) cells for 1 week. Significant increases in the following hematopoietic parameters were detected when comparing co‐cultures of fresh OBs to co‐cultures containing OBs cultured for 1, 2, or 3 weeks: total hematopoietic cell number (up to a 3.4‐fold increase), total colony forming unit (CFU) number in LSK progeny (up to an 18.1‐fold increase), and percentage of Lin‐Sca1+ cells (up to a 31.8‐fold increase). Importantly, these studies were corroborated by in vivo reconstitution studies in which LSK cells maintained in fresh OB co‐cultures supported a significantly higher level of chimerism than cells maintained in co‐cultures containing 3‐week OBs. Characterization of OBs cultured for 1, 2, or 3 weeks with real‐time PCR and functional mineralization assays showed that OB maturation increased with culture duration but was not affected by the presence of LSK cells in culture. Linear regression analyses of multiple parameters measured in these studies show that fresh, most likely more immature OBs better promote hematopoietic expansion and function than cultured, presumably more mature OBs and suggest that the hematopoiesis‐enhancing activity is mediated by cells present in fresh OB cultures de novo.

Pyk2 regulates megakaryocyte-induced increases in osteoblast number and bone formation

Preclinical and clinical evidence from megakaryocyte (MK)‐related diseases suggests that MKs play a significant role in maintaining bone homeostasis. Findings from our laboratories reveal that MKs significantly increase osteoblast (OB) number through direct MK‐OB contact and the activation of integrins. We, therefore, examined the role of Pyk2, a tyrosine kinase known to be regulated downstream of integrins, in the MK‐mediated enhancement of OBs. When OBs were co‐cultured with MKs, total Pyk2 levels in OBs were significantly enhanced primarily because of increased Pyk2 gene transcription. Additionally, p53 and Mdm2 were both decreased in OBs upon MK stimulation, which would be permissive of cell cycle entry. We then demonstrated that OB number was markedly reduced when Pyk2−/− OBs, as opposed to wild‐type (WT) OBs, were co‐cultured with MKs. We also determined that MKs inhibit OB differentiation in the presence and absence of Pyk2 expression. Finally, given that MK‐replete spleen cells from GATA‐1–deficient mice can robustly stimulate OB proliferation and bone formation in WT mice, we adoptively transferred spleen cells from these mice into Pyk2−/− recipient mice. Importantly, GATA‐1–deficient spleen cells failed to stimulate an increase in bone formation in Pyk2−/− mice, suggesting in vivo the important role of Pyk2 in the MK‐induced increase in bone volume. Further understanding of the signaling pathways involved in the MK‐mediated enhancement of OB number and bone formation will facilitate the development of novel anabolic therapies to treat bone loss diseases.

Signaling pathways involved in megakaryocyte-mediated proliferation of osteoblast lineage cells

Recent studies suggest that megakaryocytes (MKs) may play a significant role in skeletal homeostasis, as evident by the occurrence of osteosclerosis in multiple MK related diseases (Lennert et al., 1975; Thiele et al., 1999; Chagraoui et al., 2006). We previously reported a novel interaction whereby MKs enhanced proliferation of osteoblast lineage/osteoprogenitor cells (OBs) by a mechanism requiring direct cell–cell contact. However, the signal transduction pathways and the downstream effector molecules involved in this process have not been characterized. Here we show that MKs contact with OBs, via beta1 integrin, activate the p38/MAPKAPK2/p90RSK kinase cascade in the bone cells, which causes Mdm2 to neutralizes p53/Rb‐mediated check point and allows progression through the G1/S. Interestingly, activation of MAPK (ERK1/2) and AKT, collateral pathways that regulate the cell cycle, remained unchanged with MK stimulation of OBs. The MK‐to‐OB signaling ultimately results in significant increases in the expression of c‐fos and cyclin A, necessary for sustaining the OB proliferation. Overall, our findings show that OBs respond to the presence of MKs, in part, via an integrin‐mediated signaling mechanism, activating a novel response axis that de‐represses cell cycle activity. Understanding the mechanisms by which MKs enhance OB proliferation will facilitate the development of novel anabolic therapies to treat bone loss associated with osteoporosis and other bone‐related diseases. J. Cell. Physiol. 230: 578–586, 2015.

GATA-1 deficiency rescues trabecular but not cortical bone in OPG deficient mice.

GATA‐1low/low mice have an increase in megakaryocytes (MKs) and trabecular bone. The latter is thought to result from MKs directly stimulating osteoblastic bone formation while simultaneously inhibiting osteoclastogenesis. Osteoprotegerin (OPG) is known to inhibit osteoclastogenesis and OPG−/− mice have reduced trabecular and cortical bone due to increased osteoclastogenesis. Interestingly, GATA‐1low/low mice have increased OPG levels. Here, we sought to determine whether GATA‐1 knockdown in OPG−/− mice could rescue the observed osteoporotic bone phenotype. GATA‐1low/low mice were bred with OPG−/− mice and bone phenotype assessed. GATA‐1low/low × OPG−/− mice have increased cortical bone porosity, similar to OPG−/− mice. Both OPG−/− and GATA‐1low/low × OPG−/− mice, were found to have increased osteoclasts localized to cortical bone, possibly producing the observed elevated porosity. Biomechanical assessment indicates that OPG−/− and GATA‐1low/low × OPG−/− femurs are weaker and less stiff than C57BL/6 or GATA‐1low/low femurs. Notably, GATA‐1low/low × OPG−/− mice had trabecular bone parameters that were not different from C57BL/6 values, suggesting that GATA‐1 deficiency can partially rescue the trabecular bone loss observed with OPG deficiency. The fact that GATA‐1 deficiency appears to be able to partially rescue the trabecular, but not the cortical bone phenotype suggests that MKs can locally enhance trabecular bone volume, but that MK secreted factors cannot access cortical bone sufficiently to inhibit osteoclastogenesis or that OPG itself is required to inhibit osteoclastogenesis in cortical bone. J. Cell. Physiol. 230: 783–790, 2015.

Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration via Distinct and Overlapping Mechanisms

Osteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline‐rich tyrosine kinase Pyk2 (Pyk2‐KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet‐producing cells, also promote osteoblast proliferation in vitro and bone‐formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2‐KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein‐scaffolding functions, we expressed the phosphorylation‐mutant (Pyk2Y402F) and kinase‐mutant (Pyk2K457A) in Pyk2‐KO osteoblasts. Both Pyk2Y402F and Pyk2K457A reduced ALP activity, whereas only kinase‐inactive Pyk2K457A inhibited Pyk2‐KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co‐culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2‐KO osteoblasts exhibited increased migration compared to wild‐type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2‐dependent and independent signaling pathways. Novel drugs that distinguish between the kinase‐dependent or protein‐scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone‐related diseases. J. Cell. Biochem. 117: 1396–1406, 2016.

A Novel Role for Thrombopoietin in Regulating Osteoclast Development in Humans and Mice

Emerging data suggest that megakaryocytes (MKs) play a significant role in skeletal homeostasis. Indeed, osteosclerosis observed in several MK‐related disorders may be a result of increased numbers of MKs. In support of this idea, we have previously demonstrated that MKs increase osteoblast (OB) proliferation by a direct cell–cell contact mechanism and that MKs also inhibit osteoclast (OC) formation. As MKs and OCs are derived from the same hematopoietic precursor, in these osteoclastogenesis studies we examined the role of the main MK growth factor, thrombopoietin (TPO) on OC formation and bone resorption. Here we show that TPO directly increases OC formation and differentiation in vitro. Specifically, we demonstrate the TPO receptor (c‐mpl or CD110) is expressed on cells of the OC lineage, c‐mpl is required for TPO to enhance OC formation in vitro, and TPO activates the mitogen‐activated protein kinases, Janus kinase/signal transducer and activator of transcription, and nuclear factor‐kappaB signaling pathways, but does not activate the PI3K/AKT pathway. Further, we found TPO enhances OC resorption in CD14+CD110+ human OC progenitors derived from peripheral blood mononuclear cells, and further separating OC progenitors based on CD110 expression enriches for mature OC development. The regulation of OCs by TPO highlights a novel therapeutic target for bone loss diseases and may be important to consider in the numerous hematologic disorders associated with alterations in TPO/c‐mpl signaling as well as in patients suffering from bone disorders. J. Cell. Physiol. 230: 2142–2151, 2015.