Sex-dependent regulation of vertebrate somatic growth and aging by germ cells.

The function of germ cells in somatic growth and aging has been demonstrated in invertebrate models but remains unclear in vertebrates. We demonstrated sex-dependent somatic regulation by germ cells in the short-lived vertebrate model Nothobranchius furzeri. In females, germ cell removal shortened life span, decreased estrogen, and increased insulin-like growth factor 1 (IGF-1) signaling. In contrast, germ cell removal in males improved their health with increased vitamin D signaling. Body size increased in both sexes but was caused by different signaling pathways, i.e., IGF-1 and vitamin D in females and males, respectively. Thus, vertebrate germ cells regulate somatic growth and aging through different pathways of the endocrine system, depending on the sex, which may underlie the sexual difference in reproductive strategies.

Osteoblast-derived vesicles induce a switch from bone-formation to bone-resorption in vivo.

Bone metabolism is regulated by the cooperative activity between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the mechanisms mediating the switch between the osteoblastic and osteoclastic phases have not been fully elucidated. Here, we identify a specific subset of mature osteoblast-derived extracellular vesicles that inhibit bone formation and enhance osteoclastogenesis. Intravital imaging reveals that mature osteoblasts secrete and capture extracellular vesicles, referred to as small osteoblast vesicles (SOVs). Co-culture experiments demonstrate that SOVs suppress osteoblast differentiation and enhance the expression of receptor activator of NF-κB ligand, thereby inducing osteoclast differentiation. We also elucidate that the SOV-enriched microRNA miR-143 inhibits Runt-related transcription factor 2, a master regulator of osteoblastogenesis, by targeting the mRNA expression of its dimerization partner, core-binding factor ß. In summary, we identify SOVs as a mode of cell-to-cell communication, controlling the dynamic transition from bone-forming to bone-resorbing phases in vivo.

SLPI is a critical mediator that controls PTH-induced bone formation.

Osteoclastic bone resorption and osteoblastic bone formation/replenishment are closely coupled in bone metabolism. Anabolic parathyroid hormone (PTH), which is commonly used for treating osteoporosis, shifts the balance from osteoclastic to osteoblastic, although it is unclear how these cells are coordinately regulated by PTH. Here, we identify a serine protease inhibitor, secretory leukocyte protease inhibitor (SLPI), as a critical mediator that is involved in the PTH-mediated shift to the osteoblastic phase. Slpi is highly upregulated in osteoblasts by PTH, while genetic ablation of Slpi severely impairs PTH-induced bone formation. Slpi induction in osteoblasts enhances its differentiation, and increases osteoblast-osteoclast contact, thereby suppressing osteoclastic function. Intravital bone imaging reveals that the PTH-mediated association between osteoblasts and osteoclasts is disrupted in the absence of SLPI. Collectively, these results demonstrate that SLPI regulates the communication between osteoblasts and osteoclasts to promote PTH-induced bone anabolism.

[In vivo imaging of osteoblasts.]

Osteoblasts are bone-forming cells which produce bone matrix proteins and control calcium and mineral deposition. It is still unknown how osteoblasts work through the processes actually, because most of bone researches use static images of sections. Recently developed intravital imaging system with multiphoton microscopy can enable dynamic observation of living individual cells without making sections. This technique has so high-resolution in the Z direction that it is particularly useful for observation of deep tissues like bones. Here we introduce our data about osteoblast live cell imaging in vivo and in vitro including intravital imaging and summarize the latest reports about it.

Direct cell-cell contact between mature osteoblasts and osteoclasts dynamically controls their functions in vivo.

Bone homeostasis is regulated by communication between bone-forming mature osteoblasts (mOBs) and bone-resorptive mature osteoclasts (mOCs). However, the spatial-temporal relationship and mode of interaction in vivo remain elusive. Here we show, by using an intravital imaging technique, that mOB and mOC functions are regulated via direct cell-cell contact between these cell types. The mOBs and mOCs mainly occupy discrete territories in the steady state, although direct cell-cell contact is detected in spatiotemporally limited areas. In addition, a pH-sensing fluorescence probe reveals that mOCs secrete protons for bone resorption when they are not in contact with mOBs, whereas mOCs contacting mOBs are non-resorptive, suggesting that mOBs can inhibit bone resorption by direct contact. Intermittent administration of parathyroid hormone causes bone anabolic effects, which lead to a mixed distribution of mOBs and mOCs, and increase cell-cell contact. This study reveals spatiotemporal intercellular interactions between mOBs and mOCs affecting bone homeostasis in vivo.

Intercellular Communication between Keratinocytes and Fibroblasts Induces Local Osteoclast Differentiation: a Mechanism Underlying Cholesteatoma-Induced Bone Destruction.

Bone homeostasis is maintained by a balance in activity between bone-resorbing osteoclasts and bone-forming osteoblasts. Shifting the balance toward bone resorption causes osteolytic bone diseases such as rheumatoid arthritis and periodontitis. Osteoclast differentiation is regulated by receptor activator of nuclear factor κB ligand (RANKL), which, under some pathological conditions, is produced by T and B lymphocytes and synoviocytes. However, the mechanism underlying bone destruction in other diseases is little understood. Bone destruction caused by cholesteatoma, an epidermal cyst in the middle ear resulting from hyperproliferation of keratinizing squamous epithelium, can lead to lethal complications. In this study, we succeeded in generating a model for cholesteatoma, epidermal cyst-like tissue, which has the potential for inducing osteoclastogenesis in mice. Furthermore, an in vitro coculture system composed of keratinocytes, fibroblasts, and osteoclast precursors was used to demonstrate that keratinocytes stimulate osteoclast differentiation through the induction of RANKL in fibroblasts. Thus, this study demonstrates that intercellular communication between keratinocytes and fibroblasts is involved in the differentiation and function of osteoclasts, which may provide the molecular basis of a new therapeutic strategy for cholesteatoma-induced bone destruction.

Artery-dominant free jejunal transfer.

Although the supercharge (additional microvascular anastomosis) technique is often used in pedicled transfer of parts of the gastrointestinal tract, this is rarely performed during free jejunal transfer (FJT). The differences in blood circulation and outcomes between the usual single pedicle flap and a double pedicle flap are not well known. Therefore, we evaluated the effect of an additional arterial anastomosis in FJT. The FJT was performed using one venous and two arterial anastomoses after hypopharyngeal cancer ablation. To assess the effects of an arterial supercharge, blood-gas analysis, including the venous partial pressure of oxygen (pO(2)) and partial pressure of carbon dioxide (pCO(2)), was performed on samples drawn thrice from the jejunal vein: before harvest, after the anastomosis of a paired artery and vein and after an additional arterial anastomosis. The result revealed that the venous pO(2) was elevated by the additional arterial anastomosis, compared with the two other measuring times (P=0.04). The venous pCO(2) did not show significant changes. By being given a dominant artery, a jejunal flap can develop a physiological circulatory environment and can establish nutritional pathways without adverse effects.

Double vascular anastomosis for safer free jejunal transfer in unfavorable conditions.

Free jejunum was transferred to a patient with recurrent hypopharyngeal carcinoma under unfavorable cervical conditions, caused by prior therapeutic chemoradiotherapy for hypopharyngeal carcinoma and gastric pull-up with cervical leak, resulting from treatment for thoracic esophageal cancer. The cervical recipient vessels were buried in extensively scarred fibrous tissues, so they were thought to be less reliable. Because postoperative vascular occlusion was anticipated, in addition to the ordinary single vascular anastomosis to the damaged cervical vessels, secondary vascular anastomosis to the healthy chest vessels was performed. We designed the graft to have double vascular pedicles that communicated with each other through arcade vessels. This made it possible to anastomose doubly to an intact thoracoacromial artery in the chest by elongating the vascular pedicles of the mesentery without the need for an interpositional vein graft, in addition to ordinary anastomosis in the damaged neck. The flap is nourished by the vessels from two different origins (carotid and axillary arteries, internal jugular and axillary veins) at two different places (damaged and healthy areas). This method increases the quantity of feeding vessels while improving the quality of the recipient vessels within the local area and flow sources. It is one treatment option when conditions are unfavorable for safer jejunal transfer.