Requirement of Hyaluronan Synthase-2 in Craniofacial and Palate Development.

Cleft palate is a common major birth defect resulting from disruption of palatal shelf growth, elevation, or fusion during fetal palatogenesis. Whereas the molecular mechanism controlling palatal shelf elevation is not well understood, a prevailing hypothesis is that region-specific accumulation of hyaluronan, a predominant extracellular glycosaminoglycan in developing palatal mesenchyme, plays a major role in palatal shelf elevation. However, direct genetic evidence for a requirement of hyaluronan in palate development is still lacking. In this study, we show that Has2, 1 of 3 hyaluronan synthases in mammals, plays a major role in hyaluronan synthesis in the neural crest-derived craniofacial mesenchyme during palatogenesis in mice. We analyzed developmental defects caused by tissue-specific inactivation of Has2 throughout the cranial neural crest lineage or specifically in developing palatal or mandibular mesenchyme, respectively, using Wnt1-Cre, Osr2-Cre, and Hand2-Cre transgenic mice. Inactivation of Has2 either throughout the neural crest lineage or specifically in the developing palatal mesenchyme caused reduced palatal shelf size and increased palatal mesenchyme cell density prior to the time of normal palatal shelf elevation. Whereas both Has2f/f;Wnt1-Cre and Has2f/f;Osr2-Cre mutant mice exhibit cleft palate at complete penetrance, the Has2f/f; Wnt1-Cre fetuses showed dramatically reduced mandible size and complete failure of palatal shelf elevation, whereas Has2f/f;Osr2-Cre fetuses had normal mandibles and delayed palatal shelf elevation. All Has2f/f;Hand2-Cre pups showed reduced mandible size and about 50% of them had cleft palate with disruption of palatal shelf elevation. Results from explant culture assays indicate that disruption of palatal shelf elevation in Has2f/f;Hand2-Cre mutant fetuses resulted from physical obstruction by the malformed mandible and tongue. Together, these data indicate that hyaluronan plays a crucial intrinsic role in palatal shelf expansion and timely reorientation to the horizontal position above the tongue as well as an important role in mandibular morphogenesis that secondarily affects palatal shelf elevation.

Activin/Smad2 and Wnt/ß-catenin up-regulate HAS2 and ALDH3A2 to facilitate mesendoderm differentiation of human embryonic stem cells.

Activin and Wnt signaling are necessary and sufficient for mesendoderm (ME) differentiation of human embryonic stem cells (ESCs). In this study, we report that during ME differentiation induced by Activin and Wnt, Activin/Smad2 induces a decrease of the repressive histone modification of H3K27me3 by promoting the proteasome-dependent degradation of enhancer of zeste 2 polycomb (EZH2)-repressive complex 2 subunit. As a result, recruitment of the forkhead protein FOXH1 on open chromatin regions integrates the signals of Activin/Smad2 and Wnt/ß-catenin to activate the expression of the ME genes including HAS2 and ALDH3A2 Consistently, H3K27me3 decrease is enriched on open chromatin around regulatory regions. Furthermore, knockdown of HAS2 or ALDH3A2 greatly attenuates ME differentiation. These findings unveil a pathway from extracellular signals to epigenetic modification-mediated gene activation during ME commitment.

Knockout of hyaluronan synthase 1, but not 3, impairs formation of the retrocalcaneal bursa.

Hyaluronan (HA), a high molecular weight non-sulfated glycosaminoglycan, is an integral component of the extracellular matrix of developing and mature connective tissues including tendon. There are few published reports quantifying HA content during tendon growth and maturation, or detailing its effects on the mechanical properties of the tendon extracellular matrix. Therefore, the goal of the current study was to examine the role of HA synthesis during post-natal skeletal growth and maturation, and its influence on tendon structure and biomechanical function. For this purpose, the morphological, biochemical, and mechanical properties of Achilles tendons from wild type (WT) and hyaluronan synthase 1 and 3 deficient mouse strains (Has1-/- (Has1KO), Has3-/- (Has3KO), and Has1-/- 3-/- (Has1/3KO)) were determined at 4, 8, and 12 weeks of age. Overall, HAS-deficient mice did not show any marked differences from WT mice in Achilles tendon morphology or in the HA and chondroitin/dermatan sulfate (CS/DS) contents. However, HAS1-deficiency (in the single or Has1/3 double KO) impeded post-natal formation of the retrocalcaneal bursa, implicating HAS1 in regulating HA metabolism by cells lining the bursal cavity. Together, these data suggest that HA metabolism via HAS1 and HAS3 does not markedly influence the extracellular matrix structure or function of the tendon body, but plays a role in the formation/maintenance of peritendinous bursa. Additional studies are warranted to elucidate the relationship of HA and CS/DS metabolism to tendon healing and repair in vivo. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2622-2632, 2018.

Pirfenidone reduces subchondral bone loss and fibrosis after murine knee cartilage injury.

Pirfenidone is an anti-inflammatory and anti-fibrotic drug that has shown efficacy in lung and kidney fibrosis. Because inflammation and fibrosis have been linked to the progression of osteoarthritis, we investigated the effects of oral Pirfenidone in a mouse model of cartilage injury, which results in chronic inflammation and joint-wide fibrosis in mice that lack hyaluronan synthase 1 (Has1-/- ) in comparison to wild-type. Femoral cartilage was surgically injured in wild-type and Has1-/- mice, and Pirfenidone was administered in food starting after 3 days. At 4 weeks, Pirfenidone reduced the appearance, on micro-computed tomography, of pitting in subchondral bone at, and cortical bone surrounding, the site of cartilage injury. This corresponded with a reduction in fibrotic tissue deposits as observed with gross joint surface photography. Pirfenidone resulted in significant recovery of trabecular bone parameters affected by joint injury in Has1-/- mice, although the effect in wild-type was less pronounced. Pirfenidone also increased Safranin-O staining of growth plate cartilage after cartilage injury and sham operation in both genotypes. Taken together with the expression of selected extracellular matrix, inflammation, and fibrosis genes, these results indicate that Pirfenidone may confer chondrogenic and bone-protective effects, although the well-known anti-fibrotic effects of Pirfenidone may occur earlier in the wound-healing response than the time point examined in this study. Further investigations to identify the specific cell populations in the joint and signaling pathways that are responsive to Pirfenidone are warranted, as Pirfenidone and other anti-fibrotic drugs may encourage tissue repair and prevent progression of post-traumatic osteoarthritis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:365-376, 2018.

Histochemical structure and immunolocalisation of the hyaluronan system in the dromedary oviduct.

We investigated the local modulation of some histochemical properties of oviducts of the dromedary (Camelus dromedarius), focusing on the immnolocalisation of hyaluronic acid (HA) synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL1) and the HA receptor CD44 in the ampulla and isthmus. Abundant acidic mucopolysaccharides (glycosaminoglycans) were detected by Alcian blue staining along the luminal surface of both ciliated and non-ciliated epithelial cells (LE). Staining for HAS2 was higher in the primary epithelial folds of the ampulla compared with the isthmus, especially in secretory cells, adluminal epithelial surface and supranuclear cell domain. HAS3 staining was stronger in the LE of the isthmus than ampulla. HYAL2 was detected in the LE in the ampulla and isthmus and was more intense in the adluminal projections of secretory cells. HYAL1 was weakly detected in the LE with no difference between the ampulla and isthmus. Strong CD44 immunostaining was present in the LE of the ampulla and isthmus. CD44 staining was higher in secretory cells than in ciliated epithelial cells and was higher in the supranuclear region than the basal region of the cytoplasm. In conclusion, we provide evidence that HA synthesis and turnover occur in the camel oviduct. Differences in HAS2 and HAS3 expression suggest regional differences in the molecular size of HA secreted in oviductal fluid that may influence oviduct-gamete interaction in the camel.