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Long non‐coding RNA DLX6 antisense RNA 1 (DLX6‐AS1) lists a critical position in thyroid carcinoma (TC) development. However, the overall comprehension about DLX6‐AS1, microRNA (miR)‐193b‐3p and homeobox A1 (HOXA1) in TC is not thoroughly enough. Concerning to this, this work is pivoted on DLX6‐AS1/miR‐193b‐3p/HOXA1 axis in TC cell growth and autophagy. TC tissues and adjacent normal thyroid tissues were collected, in which expression of DLX6‐AS1, miR‐193b‐3p and HOXA1 was tested, together with their interactions. TC cells were transfected with DLX6‐AS1/miR‐193b‐3p‐related oligonucleotides or plasmids to test cell growth and autophagy. Tumorigenesis in nude mice was observed. DLX6‐AS1 and HOXA1 were up‐regulated, and miR‐193b‐3p was down‐regulated in TC. Depleted DLX6‐AS1 or restored miR‐193b‐3p disturbed cell growth and promoted autophagy. DLX6‐AS1 targeted miR‐193b‐3p and positively regulated HOXA1. miR‐193b‐3p inhibition mitigated the impaired tumorigenesis induced by down‐regulated DLX6‐AS1. Tumorigenesis in nude mice was consistent with that in cells. It is clear that DLX6‐AS1 depletion hinders TC cell growth and promotes autophagy via up‐regulating miR‐193b‐3p and down‐regulating HOXA1.

RATIONALE: Cardiotoxic β(1) adrenergic receptor (β(1)AR)-CaMKII signaling is a major and critical feature associated with development of heart failure. Synapse-associated protein 97 (SAP97) is a multi-functional scaffold protein that binds directly to the C-terminus of β(1)AR and organizes a receptor signalosome. OBJECTIVE: We aim to elucidate the dynamics of β(1)AR-SAP97 signalosome and its potential role in chronic cardiotoxic β(1)AR-CaMKII signaling that contributes to development of heart failure. METHODS AND RESULTS: The integrity of cardiac β(1)AR-SAP97 complex was examined in heart failure. Cardiac specific deletion of SAP97 was developed to examine β(1)AR signaling in ageing mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β(1)AR-SAP97 signaling complex is reduced in heart failure. Cardiac specific deletion of SAP97 yields an ageing-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA-dependent association of β(1)AR with arrestin2 and CaMKII and turns on an Epac-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 is necessary to promote agonist-induced dissociation of SAP97 from β(1)AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β(1)AR-SAP97 complex and increases in CaMKII activity in hearts. CONCLUSIONS: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac β(1)AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy.

Rationale: Cardiotoxic β 1 adrenergic receptor (β 1 AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of β 1 AR and organizes a receptor signalosome. Objective: We aim to elucidate the dynamics of β 1 AR-SAP97 signalosome and its potential role in chronic cardiotoxic β 1 AR-CaMKII signaling that contributes to development of heart failure. Methods and Results: The integrity of cardiac β 1 AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine β 1 AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β 1 AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of β 1 AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from β 1 AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β 1 AR-SAP97 complex and increases in CaMKII activity in hearts. Conclusions: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac β 1 AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

Patients with essential hypertension undergo endothelial dysfunction, particularly in the conduit arteries. Cilostazol, a type III phosphodiesterase inhibitor, serves a role in the inhibition of platelet aggregation and it is widely used in the treatment of peripheral vascular diseases. Previous studies have suggested that cilostazol suppresses endothelial dysfunction; however, it remains unknown whether cilostazol protects the endothelial function in essential hypertension. The aim of the present study was to investigate whether, and how, cilostazol suppresses angiotensin II (angII)‑induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) and Sprague Dawley rats were exposed to angII and treated with cilostazol. Endothelial cell apoptosis and function, nitric oxide and superoxide production, phosphorylation (p) of Akt, and caspase‑3 protein expression levels were investigated. AngII exposure resulted in the apoptosis of endothelial cells in vitro and in vivo. In vitro, cilostazol significantly suppressed the angII‑induced apoptosis of HUVECs; however, this effect was reduced in the presence of LY294002, a phosphoinositide 3 kinase (PI3K) inhibitor. Furthermore, cilostazol suppressed the angII‑induced p‑Akt downregulation and cleaved caspase‑3 upregulation. These effects were also alleviated by LY294002. In vivo, cilostazol suppressed the angII‑induced endothelial cell apoptosis and dysfunction. Cilostazol was also demonstrated to partially reduced the angII‑induced increase in superoxide production. The results of the present study suggested that cilostazol suppresses endothelial apoptosis and dysfunction by modulating the PI3K/Akt pathway.

Why regeneration does not occur in mammals remains elusive. In lower vertebrates, epimorphic regeneration of the limb is directed by the wound epidermis, which controls blastema formation to promote regrowth of the appendage. Herein, we report that knockout (KO) or inhibition of Apoptosis Signal-regulated Kinase-1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5), after full thickness ear punch in mice prolongs keratinocyte activation within the wound epidermis and promotes regeneration of auricular cartilage. Histological analysis showed the ASK1 KO ears displayed enhanced protein markers associated with blastema formation, hole closure and regeneration of auricular cartilage. At seven days after punch, the wound epidermis morphology was markedly different in the KO, showing a thickened stratum corneum with rounded cell morphology and a reduction of both the granular cell layer and decreased expression of filament aggregating protein. In addition, cytokeratin 6 was expressed in the stratum spinosum and granulosum. Topical application of inhibitors of ASK1 (NQDI-1), the upstream ASK1 activator, calcium activated mitogen kinase 2 (KN93), or the downstream target, c-Jun N-terminal kinase (SP600125) also resulted in enhanced regeneration; whereas inhibition of the other downstream target, the p38 α/β isoforms, (SB203580) had no effect. The results of this investigation indicate ASK1 inhibition prolongs keratinocyte and blastemal cell activation leading to ear regeneration.

Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has clinical potential to treat fractures or to slow osteoarthritic progression by enhancing chondrocyte survival and slowing hypertrophy.