Mark C. Horowitz

Adipocyte Lineage Cells Contribute to the Skin Stem Cell Niche to Drive Hair Cycling

In mammalian skin, multiple types of resident cells are required to create a functional tissue and support tissue homeostasis and regeneration. The cells that compose the epithelial stem cell niche for skin homeostasis and regeneration are not well defined. Here, we identify adipose precursor cells within the skin and demonstrate that their dynamic regeneration parallels the activation of skin stem cells. Functional analysis of adipocyte lineage cells in mice with defects in adipogenesis and in transplantation experiments revealed that intradermal adipocyte lineage cells are necessary and sufficient to drive follicular stem cell activation. Furthermore, we implicate PDGF expression by immature adipocyte cells in the regulation of follicular stem cell activity. These data highlight adipogenic cells as skin niche cells that positively regulate skin stem cell activity, and suggest that adipocyte lineage cells may alter epithelial stem cell function clinically.

Monoclonal antibodies reactive with human osteogenic cell surface antigens

Monoclonal antibodies (McAbs) against the surface of osteoblastic cells have been used to characterize the osteogenic lineage. In view of the paucity of probes against the surface of normal human osteogenic cells, we sought to generate McAbs which could be used for both in vivo and in vitro studies. We raised a series of McAbs against early osteoblastic cell surface antigens by immunizing mice with human mesenchymal stem cells (MSCs) that had been directed into the osteogenic lineage in vitro. After screening against the surface of osteogenic cells at various stages of differentiation in vitro, as well as evaluating in situ reactivity with human fetal limbs, we isolated three hybridoma cell lines referred to as SB-10, SB-20, and SB-21. Immunocytochemical analyses during osteogenic differentiation demonstrate that SB-10 reacts with MSCs and osteoprogenitors, but no longer reacts with cells once alkaline phosphatase (APase) is expressed. Flow cytometry documents that SB-10 is expressed on the surface of all purified, culture-expanded human MSCs, thus providing further evidence that these cells are a homogeneous population. By contrast, SB-20 and SB-21 do not react with the progenitor cells in situ, but bind to a subset of the APase-positive osteoblasts. None of these antibodies stain terminally differentiated osteocytes in sections of developing bone. Furthermore, these McAbs were not observed to react in samples from chick, rat, rabbit, canine, or bovine bone, although selected extraskeletal human tissues were immunostained. In all cell and tissue specimens examined, SB-20 immunostaining is identical to that observed with SB-21. We have used these McAbs to refine our understanding of the discrete cellular transitions that constitute the osteogenic cell lineage. We suggest a refined model for understanding osteoblast differentiation that is based on the proposition that the sequential acquisition and loss of specific cell surface molecules can be used to define positions of individual cells within the osteogenic cell lineage.

Control of osteoclastogenesis and bone resorption by members of the TNF family of receptors and ligands.

Skeletal mass is maintained by a balance between cells which resorb bone (osteoclasts) and cells which form bone (osteoblasts). Bone development and growth is an on-going, life-long process. Bone is formed during embryonic life, grows rapidly through childhood, and peaks around 20 years of age (formation exceeds resorption). For humans the skeleton then enters a long period, approximately 40 years, when bone mass remains relatively stable. Skeletal turnover continues but the net effect of resorption and formation on bone mass is zero. For women this ends when they enter menopause and similar bone loss occurs for men, but later in life. These opposite functions are coupled, resorption precedes formation, and osteoblasts, or their precursors, stromal cells, regulate osteoclast formation and activity. Until recently, the molecular nature of this regulation, was poorly understood. However, recent observations have identified members of the TNF family of ligands and receptors as critical regulators of osteoclastogenesis. Osteoprotegerin (OPG) a decoy receptor was first identified. Its ligand, receptor activator of nuclear factor-kappaB ligand (RANKL), was quickly found, and shown to be expressed on stromal cells and osteoblasts. Its cognate receptor, RANK, was found to be expressed in high levels on osteoclast precursors. The interaction between RANKL and RANK was shown to be required for osteoclast formation. These observations have provided a molecular understanding of the coupling between osteoclastic bone resorption and osteoblastic bone formation. Moreover, they provide a framework on which to base a clear understanding of normal (e.g. postmenopausal osteoporosis and age associated bone loss) and pathologic skeletal changes (e.g. osteopetrosis, glucocorticoid-induced osteoporosis, periodontal disease, bone metastases, Pagets disease, hyperparathyroidism, and rheumatoid arthritis).

TOPGAL mice show that the canonical Wnt signaling pathway is active during bone development and growth and is activated by mechanical loading in vitro.

We identified cellular targets of canonical Wnt signaling within the skeleton, which included chondrocytes, osteoblasts, and osteocytes in growing bone, but only osteocytes and chondrocytes in the mature skeleton. Mechanical deformation induced Wnt signaling in osteoblasts in vitro.

Marrow fat and bone--new perspectives.

CONTEXT There is growing interest in the relationship between bone mineral density, bone strength, and fat depots. Marrow adipose tissue, a well-established component of the marrow environment, is metabolically distinct from peripheral fat depots, but its functional significance is unknown. OBJECTIVE In this review, we discuss animal and human data linking the marrow adipose tissue depot to parameters of bone density and integrity as well as the potential significance of marrow adipose tissue in metabolic diseases associated with bone loss, including type 1 diabetes mellitus and anorexia nervosa. Potential hormonal determinants of marrow adipose tissue are also discussed. CONCLUSIONS We conclude that whereas most animal and human data demonstrate an inverse association between marrow adipose tissue and measures of bone density and strength, understanding the functional significance of marrow adipose tissue and its hormonal determinants will be critical to better understanding its role in skeletal integrity and the role of marrow adipose tissue in the pathophysiology of bone loss.

Bone Marrow Adipose Tissue Is an Endocrine Organ that Contributes to Increased Circulating Adiponectin during Caloric Restriction

The adipocyte-derived hormone adiponectin promotes metabolic and cardiovascular health. Circulating adiponectin increases in lean states such as caloric restriction (CR), but the reasons for this paradox remain unclear. Unlike white adipose tissue (WAT), bone marrow adipose tissue (MAT) increases during CR, and both MAT and serum adiponectin increase in many other clinical conditions. Thus, we investigated whether MAT contributes to circulating adiponectin. We find that adiponectin secretion is greater from MAT than WAT. Notably, specific inhibition of MAT formation in mice results in decreased circulating adiponectin during CR despite unaltered adiponectin expression in WAT. Inhibiting MAT formation also alters skeletal muscle adaptation to CR, suggesting that MAT exerts systemic effects. Finally, we reveal that both MAT and serum adiponectin increase during cancer therapy in humans. These observations identify MAT as an endocrine organ that contributes significantly to increased serum adiponectin during CR and perhaps in other adverse states.

Megakaryocyte-osteoblast interaction revealed in mice deficient in transcription factors GATA-1 and NF-E2

Mice deficient in GATA‐1 or NF‐E2 have a 200–300% increase in bone volume and formation parameters. Osteoblasts and osteoclasts generated in vitro from mutant and control animals were similar in number and function. Osteoblast proliferation increased up to 6‐fold when cultured with megakaryocytes. A megakaryocyte‐osteoblast interaction plays a role in the increased bone formation in these mice.

Parathyroid hormone and lipopolysaccharide induce murine osteoblast-like cells to secrete a cytokine indistinguishable from granulocyte-macrophage colony-stimulating factor.

Osteoblasts are the cells responsible for the secretion of collagen and ultimately the formation of new bone. These cells have also been shown to regulate osteoclast activity by the secretion of cytokines, which remain to be defined. In an attempt to identify these unknown cytokines, we have induced primary murine osteoblasts with two bone active agents, parathyroid hormone (PTH) and lipopolysaccharide (LPS) and analyzed the conditioned media (CM) for the presence of specific cytokines. Analysis of the CM was accomplished by functional, biochemical, and serological techniques. The data indicate that both PTH and LPS are capable of inducing the osteoblasts to secrete a cytokine, which by all of the techniques used, is indistinguishable from granulocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of GM-CSF is not constitutive and requires active induction. Production of the cytokine is dependent on the dose of PTH or LPS added. It has been demonstrated that the addition of GM-CSF to bone marrow cultures results in the formation of increased numbers of osteoclasts. Therefore, these data suggest that osteoblasts not only participate in bone remodeling by formation of new matrix but may regulate osteoclast activity indirectly by their ability to regulate hematopoiesis.

A circadian-regulated gene, Nocturnin, promotes adipogenesis by stimulating PPAR-γ nuclear translocation

Nocturnin (NOC) is a circadian-regulated protein related to the yeast family of transcription factors involved in the cellular response to nutrient status. In mammals, NOC functions as a deadenylase but lacks a transcriptional activation domain. It is highly expressed in bone-marrow stromal cells (BMSCs), hepatocytes, and adipocytes. In BMSCs exposed to the PPAR-γ (peroxisome proliferator-activated receptor-γ) agonist rosiglitazone, Noc expression was enhanced 30-fold. Previously, we reported that Noc−/− mice had low body temperature, were protected from diet-induced obesity, and most importantly exhibited absence of Pparg circadian rhythmicity on a high-fat diet. Consistent with its role in influencing BMSCs allocation, Noc−/− mice have reduced bone marrow adiposity and high bone mass. In that same vein, NOC overexpression enhances adipogenesis in 3T3-L1 cells but negatively regulates osteogenesis in MC3T3-E1 cells. NOC and a mutated form, which lacks deadenylase activity, bind to PPAR-γ and markedly enhance PPAR-γ transcriptional activity. Both WT and mutant NOC facilitate nuclear translocation of PPAR-γ. Importantly, NOC-mediated nuclear translocation of PPAR-γ is blocked by a short peptide fragment of NOC that inhibits its physical interaction with PPAR-γ. The inhibitory effect of this NOC-peptide was partially reversed by rosiglitazone, suggesting that effect of NOC on PPAR-γ nuclear translocation may be independent of ligand-mediated PPAR-γ activation. In sum, Noc plays a unique role in the regulation of mesenchymal stem-cell lineage allocation by modulating PPAR-γ activity through nuclear translocation. These data illustrate a unique mechanism whereby a nutrient-responsive gene influences BMSCs differentiation, adipogenesis, and ultimately body composition.

A reciprocal regulatory interaction between megakaryocytes, bone cells, and hematopoietic stem cells

A growing body of evidence suggests that megakaryocytes (MK) or their growth factors play a role in skeletal homeostasis. MK have been shown to express and/or secrete several bone-related proteins including osteocalcin, osteonectin, bone sialoprotein, osteopontin, bone morphogenetic proteins, and osteoprotegerin. In addition, at least 3 mouse models have been described in which MK number was significantly elevated with an accompanying marked increase in bone mineral density. Mice overexpressing thrombopoietin, the major MK growth factor, have an osteosclerotic bone phenotype. Mice deficient in transcription factors GATA-1 and NF-E2, which are required for the differentiation of MK, exhibited a strikingly increased bone mass. Importantly, recent studies have demonstrated that MK can stimulate osteoblast (OB) proliferation and differentiation in vitro and that they can also inhibit osteoclast (OC) formation in vitro. These findings suggest that MK play a dual role in skeletal homeostasis by stimulating formation while simultaneously inhibiting resorption. Conversely, cells of the osteoblast lineage support hematopoiesis, including megakaryopoiesis. Postnatal hematopoiesis occurs almost solely in the bone marrow (BM), close to or on endosteal surfaces. This finding, in conjunction with the observed contact of OB with hematopoietic cells, has lead investigators to explore the molecular and cellular interactions between hematopoietic cells and cells of the OB lineage. Importantly, it has been shown that many of the cytokines that are critical for normal hematopoiesis and megakaryopoiesis are produced by OB. Indeed, culturing osteoblasts with CD34+ BM cells significantly enhances hematopoietic cell number by both enhancing the proliferation of long-term culture initiating cells and the proliferation and differentiation of MK. These data are consistent with cells in the OB lineage playing a critical role in the hematopoietic niche. Overall, these observations demonstrate the importance of MK-bone cell interactions in both skeletal homeostasis and hematopoiesis.