Manabu Kawata

Transcription factor Hes1 modulates osteoarthritis development in cooperation with calcium/calmodulin-dependent protein kinase 2

Significance Here we demonstrate that Hes1, an important target of Notch signaling, modulated pathogenesis of osteoarthritis by using Col2a1-CreERT;Hes1fl/fl mice. Adamts5 and Mmp13, catabolic enzymes that break down cartilage matrix, were induced by Hes1. Additionally, microarray analysis and ChIP-seq revealed novel Hes1 target genes, including Il6 and Il1rl1, coding a receptor for IL-33. CaMK2δ was activated during osteoarthritis development. CaMK2δ formed a protein complex with Hes1, and switched it from a transcriptional repressor to a transcriptional activator to induce cartilage catabolic factors. Notch signaling modulates skeletal formation and pathogenesis of osteoarthritis (OA) through induction of catabolic factors. Here we examined roles of Hes1, a transcription factor and important target of Notch signaling, in these processes. SRY-box containing gene 9 (Sox9)-Cre mice were mated with Hes1fl/fl mice to generate tissue-specific deletion of Hes1 from chondroprogenitor cells; this deletion caused no obvious abnormality in the perinatal period. Notably, OA development was suppressed when Hes1 was deleted from articular cartilage after skeletal growth in type II collagen (Col2a1)-CreERT;Hes1fl/fl mice. In cultured chondrocytes, Hes1 induced metallopeptidase with thrombospondin type 1 motif, 5 (Adamts5) and matrix metalloproteinase-13 (Mmp13), which are catabolic enzymes that break down cartilage matrix. ChIP-seq and luciferase assays identified Hes1-responsive regions in intronic sites of both genes; the region in the ADAMTS5 gene contained a typical consensus sequence for Hes1 binding, whereas that in the MMP13 gene did not. Additionally, microarray analysis, together with the ChIP-seq, revealed novel Hes1 target genes, including Il6 and Il1rl1, coding a receptor for IL-33. We further identified calcium/calmodulin-dependent protein kinase 2δ (CaMK2δ) as a cofactor of Hes1; CaMK2δ was activated during OA development, formed a protein complex with Hes1, and switched it from a transcriptional repressor to a transcriptional activator to induce cartilage catabolic factors. Therefore, Hes1 cooperated with CaMK2δ to modulate OA pathogenesis through induction of catabolic factors, including Adamts5, Mmp13, Il6, and Il1rl1. Our findings have contributed to further understanding of the molecular pathophysiology of OA, and may provide the basis for development of novel treatments for joint disorders.

Hyaline cartilage formation and tumorigenesis of implanted tissues derived from human induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) are a promising cell source for cartilage regenerative medicine. Meanwhile, the risk of tumorigenesis should be considered in the clinical application of human iPSCs (hiPSCs). Here, we report in vitro chondrogenic differentiation of hiPSCs and maturation of the differentiated hiPSCs through transplantation into mouse knee joints. Three hiPSC clones showed efficient chondrogenic differentiation using an established protocol for human embryonic stem cells. The differentiated hiPSCs formed hyaline cartilage tissues at 8 weeks after transplantation into the articular cartilage of NOD/SCID mouse knee joints. Although tumors were not observed during the 8 weeks after transplantation, an immature teratoma had developed in one mouse at 16 weeks. In conclusion, hiPSCs are a potent cell source for regeneration of hyaline articular cartilage. However, the risk of tumorigenesis should be managed for clinical application in the future.

Regulation of Chondrocyte Survival in Mouse Articular Cartilage by p63

Transcription factor p63, of the p53 family, regulates cell proliferation, survival, and apoptosis in various cells and tissues. This study was undertaken to examine the expression and roles of p63 transcript variants in the mouse growth plate and articular chondrocytes.

p63 Regulates Chondrocyte Survival in Articular Cartilage

Transcription factor p63, of the p53 family, regulates cell proliferation, survival, and apoptosis in various cells and tissues. This study was undertaken to examine the expression and roles of p63 transcript variants in the mouse growth plate and articular chondrocytes.

Annual trends in knee arthroplasty and tibial osteotomy: Analysis of a national database in Japan

BACKGROUND Various nationwide studies have reported differing annual trends in utilization of knee arthroplasty and tibial osteotomy. Using the Diagnosis Procedure Combination database in Japan, the present series examined annual trends and demographics in total knee arthroplasty (TKA), unicompartmental knee arthroplasty (UKA) and tibial osteotomy. METHODS All patients were identified who underwent TKA, UKA or tibial osteotomy for osteoarthritis, osteonecrosis or rheumatoid arthritis of the knee between July 2007 and March 2015. RESULTS A total of 170,433 cases of TKA, 13,209 cases of UKA and 8760 cases of tibial osteotomy were identified. The proportion of patients undergoing UKA rose from 4.0% in 2007 to 8.1% in 2014 (P<0.001), and that of tibial osteotomy from 2.6% in 2007 to 5.5% in 2014 (P<0.001); the proportion undergoing TKA fell from 93.4% in 2007 to 86.3% in 2014 (P<0.001). Between 2007 and 2014 the proportions of patients with osteonecrosis who underwent UKA and tibial osteotomy increased from 34.7% and 11.6% to 38.6% and 16.2%, respectively (P=0.001 for UKA and P=0.004 for tibial osteotomy). The proportions of patients with osteonecrosis undergoing UKA or tibial osteotomy were significantly greater than those with other diagnoses (P<0.001 for both). CONCLUSIONS The popularity of UKA and tibial osteotomy in Japan increased during the period 2007-2014 at the expense of TKA. The proportions of UKA and tibial osteotomy in patients with osteonecrosis also increased, and were larger than those in patients with other causative diseases.

Tenosynovitis with psammomatous calcification arising from the volar plate of the proximal interphalangeal joint of the finger.

To the Editor: Acute onset of pain and swelling in the distal extremities can be provoked by calcareous lesions even in the absence of metabolic disorders, such as hyperphosphatemia, and these lesions often complicate differential diagnosis for pathologists and clinicians. A few reports described cases of tenosynovitis with psammomatous calcification as repetitive trauma-associated pseudotumor originating from tendons and peritendinous soft tissues. However, no report has indicated that tenosynovitis with psammomatous calcification could develop from the volar plate. We hereby present a case of tenosynovitis with psammomatous calcification arising from the volar plate of the proximal interphalangeal (PIP) joint of the middle finger. A 35-year-old man presented with painful swelling on the palmar side of the PIP joint of the left middle finger, which appeared 3 weeks previously with no obvious trauma. He had no medical history of metabolic disorder or autoimmune disease. During physical examination, a soft tender mass of 5 mm in diameter was detected on the radiopalmar side of the PIP joint of the left middle finger. Radiographic images of this finger revealed a calcified lesion consistent with the mass (Fig. 1a,b). Blood tests, including inflammatory markers, calcium, phosphorus, and uric acid, were unremarkable. The mass was surgically excised because of severe pain refractory to analgesics. The mass was located deep in the distal volar plate of the PIP joint, and a small fraction extended into the joint space. The yellowish, soft, and relatively well-defined lesion was marginally resected. The pain disappeared immediately after the operation, and there has been no evidence of recurrence for at least 16 months. Pathological examination of the resected mass revealed a partially cystic and variably cellular lesion, with numerous psammomatous calcifications, and the accumulation of histiocytes and fibroblasts (Fig. S1a,b). High magnification microscopic examination showed that the psammomatous calcifications were surrounded by a vaguely granulomatous proliferation of mitotically active histiocytes and fibroblasts (Fig. 2a). Multinucleated giant cells were scattered throughout the mass (Fig. 2b). However, siderophages and foam cells were not detected, and there was no sign of necrosis or malignancy. These histological features were largely similar to tenosynovitis with psammomatous calcification. Immunohistochemistry for vimentin was diffusely positive (Fig. 2c). On the other hand, CD68 was positive in the multinucleated giant cells (arrows) and some of the mononuclear cells (arrowheads). (Fig. 2d). Tenosynovitis with psammomatous calcification was first proposed as a variant of idiopathic calcifying tenosynovitis in 1983 by Gravanis and Gaffney. However, few other studies have reported this entity. This clinicopathological entity is histologically characterized as a lesion containing numerous psammoma-like calcifications, surrounded by a vaguely granulomatous proliferation of mitotically active histiocytes and fibroblasts. From a clinical point of view, the tenosynovitis appears to have no association with metabolic disorders, to be far more common among women for unclear reasons, and to be recurrence-free after surgical excision. Shon and Folpe reported that their six patients presenting tenosynovitis with psammomatous calcification had occupational or sports-related backgrounds, which potentially exposed the affected area to microtrauma. Therefore, they argued that this tenosynovitis was associated with repetitive tendinous injury. The volar plate of finger PIP joints may also be subjected to repetitive microtrauma because it performs various stabilizing functions to prevent hyperextension, lateral displacement, and torsion of the joint. These studies suggest that the

Annual trends in arthroscopic meniscus surgery: Analysis of a national database in Japan

Background The importance of meniscus preservation is widely recognized. There have been a few studies describing trends in meniscectomy and meniscus repair in the United States; however, they presented differing results. We reported annual trends in meniscus surgery, using a national database in Japan. Methods We interrogated the Diagnosis Procedure Combination database, which represents approximately half of all hospital admissions in Japan. We included the patients who underwent meniscectomy and meniscus repair between July 2007 and March 2015. The diagnosis, age and sex of each patient were recorded. Results We identified 83,105 patients: 69,310 underwent meniscectomy; 13,416 underwent meniscus repair and 379 underwent both in a single admission. The proportion of patients undergoing meniscus repair rose from 7.0% in 2007 to 25.9% in 2014 (p < 0.001), while the proportion undergoing meniscectomy fell from 92.8% in 2007 to 73.3% in 2014 (p < 0.001). Among patients under 30 years old, the proportions undergoing meniscus repair or meniscectomy in 2014 were 50.3% versus 48.3%, respectively. A bimodal age distribution was observed for meniscectomy, with peaks at 10–19 years of age and 60–69 years of age, whereas most patients undergoing meniscus repair were 10–19 years of age. Conclusions We found characteristic trends where the popularity of meniscus repair increased rapidly at the expense of meniscectomy in Japan during the study period. In the last survey year, the proportion of meniscus repair exceeded that of meniscectomy in those younger than 30 years. Meniscectomy was undertaken most often in adolescents and early old age, while meniscus repair was undertaken most often in adolescents.

Different regulation of limb development by p63 transcript variants

The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.