The miR-29-3p family suppresses inflammatory osteolysis.

Osteoclasts are the cells primarily responsible for inflammation-induced bone loss, as is particularly seen in rheumatoid arthritis. Increasing evidence suggests that osteoclasts formed under homeostatic versus inflammatory conditions may differ in phenotype. While microRNA-29-3p family members (miR-29a-3p, miR-29b-3p, miR-29c-3p) promote the function of RANKL-induced osteoclasts, the role of miR-29-3p during inflammatory TNF-α-induced osteoclastogenesis is unknown. We used bulk RNA-seq, histology, qRT-PCR, reporter assays, and western blot analysis to examine bone marrow monocytic cell cultures and tissue from male mice in which the function of miR-29-3p family members was decreased by expression of a miR-29-3p tough decoy (TuD) competitive inhibitor in the myeloid lineage (LysM-cre). We found that RANKL-treated monocytic cells expressing the miR-29-3p TuD developed a hypercytokinemia/proinflammatory gene expression profile in vitro, which is associated with macrophages. These data support the concept that miR-29-3p suppresses macrophage lineage commitment and may have anti-inflammatory effects. In correlation, when miR-29-3p activity was decreased, TNF-α-induced osteoclast formation was accentuated in an in vivo model of localized osteolysis and in a cell-autonomous manner in vitro. Further, miR-29-3p targets mouse TNF receptor 1 (TNFR1/Tnfrsf1a), an evolutionarily conserved regulatory mechanism, which likely contributes to the increased TNF-α signaling sensitivity observed in the miR-29-3p decoy cells. Whereas our previous studies demonstrated that the miR-29-3p family promotes RANKL-induced bone resorption, the present work shows that miR-29-3p dampens TNF-α-induced osteoclastogenesis, indicating that miR-29-3p has pleiotropic effects in bone homeostasis and inflammatory osteolysis. Our data supports the concept that the knockdown of miR-29-3p activity could prime myeloid cells to respond to an inflammatory challenge and potentially shift lineage commitment toward macrophage, making the miR-29-3p family a potential therapeutic target for modulating inflammatory response.

Content Validity Testing of a Novel Cognitive Screen, the Gaylord Occupational Therapy Cognitive (GOT-Cog), to Improve Inpatient Occupational Therapy Treatment Planning.

IMPORTANCE: No single cognitive screen adequately captures all cognitive domains that are important for inpatient occupational therapy treatment planning. OBJECTIVE: To quantify the content validity of a novel 22-item cognitive screen, the Gaylord Occupational Therapy Cognitive (GOT-Cog) screen, developed to better inform inpatient occupational therapy treatment planning. DESIGN: Delphi-style expert panel review. SETTING: Long-term acute care hospital. PARTICIPANTS: The first panel was attended by four occupational therapists, two speech-language pathologists, one physician assistant, and two neuropsychologists; the second, by four occupational therapists, one speech-language pathologist, and one physician assistant. INTERVENTION: Each Delphi panel discussed the relevance, essentiality, and clarity of each item. After each discussion, panelists completed a content validity survey to summarize their evaluation of each item. OUTCOMES AND MEASURES: On the basis of panelists' survey responses, item- and scale-level relevance, essentiality, and clarity were quantified by calculating the respective content validity index (CVI), content validity ratio (CVR), and content clarity index (CCI). Universal agreement (UA) and κ statistics were also calculated, as appropriate. RESULTS: Upon presenting the initial 23-item instrument covering 10 cognitive domains to the first Delphi panel, several questions were added, removed, or rewritten, resulting in a 22-item instrument representing nine domains. After the second panel, several questions were again rewritten, and the domains reorganized. All scale-level metrics improved, including CVI (from 0.87 to 1.0), UA (0.52 to 1.0), CVR (0.43 to 0.94), and CCI (2.26 to 2.92). CONCLUSIONS AND RELEVANCE: GOT-Cog displays overall excellent content validity and can proceed to construct validity testing. Plain-Language Summary: By reporting on the content validity of the Gaylord Occupational Therapy Cognitive screen, this brief report begins the necessary process of evaluating the measure's overall validity and reliability.

MicroRNA-433 Dampens Glucocorticoid Receptor Signaling, Impacting Circadian Rhythm and Osteoblastic Gene Expression.

Serum glucocorticoids play a critical role in synchronizing circadian rhythm in peripheral tissues, and multiple mechanisms regulate tissue sensitivity to glucocorticoids. In the skeleton, circadian rhythm helps coordinate bone formation and resorption. Circadian rhythm is regulated through transcriptional and post-transcriptional feedback loops that include microRNAs. How microRNAs regulate circadian rhythm in bone is unexplored. We show that in mouse calvaria, miR-433 displays robust circadian rhythm, peaking just after dark. In C3H/10T1/2 cells synchronized with a pulse of dexamethasone, inhibition of miR-433 using a tough decoy altered the period and amplitude of Per2 gene expression, suggesting that miR-433 regulates rhythm. Although miR-433 does not directly target the Per2 3'-UTR, it does target two rhythmically expressed genes in calvaria, Igf1 and Hif1α. miR-433 can target the glucocorticoid receptor; however, glucocorticoid receptor protein abundance was unaffected in miR-433 decoy cells. Rather, miR-433 inhibition dramatically enhanced glucocorticoid signaling due to increased nuclear receptor translocation, activating glucocorticoid receptor transcriptional targets. Last, in calvaria of transgenic mice expressing a miR-433 decoy in osteoblastic cells (Col3.6 promoter), the amplitude of Per2 and Bmal1 mRNA rhythm was increased, confirming that miR-433 regulates circadian rhythm. miR-433 was previously shown to target Runx2, and mRNA for Runx2 and its downstream target, osteocalcin, were also increased in miR-433 decoy mouse calvaria. We hypothesize that miR-433 helps maintain circadian rhythm in osteoblasts by regulating sensitivity to glucocorticoid receptor signaling.

Implementation of a free water protocol at a long term acute care hospital.

This feasibility study aimed to trial a Free Water Protocol (FWP) for patients with thin liquid dysphagia in the Long-Term Acute Care Hospital (LTACH) setting. Patients with dysphagia are often prescribed thickened liquids to avoid or mitigate aspiration. While this clinical intervention can minimize the risk of aspiration pneumonia (PNA), it is generally not well received by patients. As such, the goal of this study was to determine if patients who knowingly aspirate thin liquids can safely tolerate thin liquid water, and if so, to what degree of benefit. The study assessed for adverse events, fluid intake, hydration status, quality of life, and overall swallow function outcomes. These measurements were taken over a 7 day trial period using inventories, lab work, clinical judgment, and observation. Ten participants were enrolled in this study with 9 having sufficient data for analysis (n = 9). No adverse events related to the FWP were observed, and patients saw improved total fluid intake (P = 0.0074), swallow-related quality of life (P = 0.0273), and overall swallow function (P = 0.0002). The results in this feasibility study allowed for the hospital wide implementation of the FWP and laid out the groundwork for future studies looking at longitudinal effects of a FWP.

Inhibition of miR-29 Activity in the Myeloid Lineage Increases Response to Calcitonin and Trabecular Bone Volume in Mice.

The miR-29-3p family (miR-29a, miR-29b, miR-29c) of microRNAs is increased during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis. In vivo, activation of a miR-29-3p tough decoy inhibitor in Cre recombinase under the control of the lysozyme 2 promoter-expressing cells (myeloid lineage) resulted in mice displaying enhanced trabecular and cortical bone volume because of decreased bone resorption. Calcitonin receptor (Calcr) is a miR-29 target that negatively regulates bone resorption. CALCR was significantly increased in RANKL-treated miR-29-decoy osteoclasts, and these cells were more responsive to the inhibitory effect of calcitonin on osteoclast formation. Further, cathepsin K (Ctsk), which is critical for resorption, was decreased in miR-29-decoy cells. CALCR is a Gs-coupled receptor and its activation raises cAMP levels. In turn, cAMP suppresses cathepsin K, and cAMP levels were increased in miR-29-decoy cells. siRNA-mediated knock-down of Calcr in miR-29 decoy osteoclasts allowed recovery of cathepsin K levels in these cells. Overall, using a novel knockin tough decoy mouse model, we identified a new role for miR-29-3p in bone homeostasis. In RANKL-driven osteoclastogenesis, as seen in normal bone remodeling, miR-29-3p promotes resorption. Consequently, inhibition of miR-29-3p activity in the myeloid lineage leads to increased trabecular and cortical bone. Further, this study documents an interrelationship between CALCR and CTSK in osteoclastic bone resorption, which is modulated by miR-29-3p.

Inhibition of miR-29-3p isoforms via tough decoy suppresses osteoblast function in homeostasis but promotes intermittent parathyroid hormone-induced bone anabolism.

miRNAs play a vital role in post-transcriptional regulation of gene expression in osteoblasts and osteoclasts, and the miR-29 family is expressed in both lineages. Using mice globally expressing a miR-29-3p tough decoy, we demonstrated a modest 30-60% decrease all three miR-29-3p isoforms: miR-29a, miR-29b, and miR-29c. While the miR-29-3p decoy did not impact osteoclast number or function, the tough decoy decreased bone formation in growing mice, which led to decreased trabecular bone volume in mature animals. These data support previous in vitro studies suggesting that miR-29-3p is a positive regulator of osteoblast differentiation. In contrast, when mice were treated with intermittent parathyroid hormone (PTH1-34), inhibition of miR-29-3p augmented the effect of PTH on cortical bone anabolism, increased bone formation rate and osteoblast surface, and increased levels of Ctnnb1/ßcatenin mRNA, which is a miR-29 target. These findings highlight differences in the mechanisms controlling basal level bone formation and bone formation induced by intermittent PTH. Overall, the global miR-29-3p tough decoy model represents a modest loss-of-function, which could be a relevant tool for assessing the possible impact of systemically administered miR-29-3p inhibitors. Our studies provide a potential rationale for co-administration of PTH1-34 and miR-29-3p inhibitors, to boost bone formation in severely affected osteoporosis patients, particularly in the cortical compartment.

MicroRNAs are Critical Regulators of Osteoclast Differentiation.

PURPOSE OF REVIEW: Our goal is to comprehensively review the most recent reports of microRNA (miRNA) regulation of osteoclastogenesis. We highlight validated miRNA-target interactions and their place in the signaling networks controlling osteoclast differentiation and function. RECENT FINDINGS: Using unbiased approaches to identify miRNAs of interest and reporter-3'UTR assays to validate interactions, recent studies have elucidated the impact of specific miRNA-mRNA interactions during in vitro osteoclastogenesis. There has been a focus on signaling mediators downstream of the RANK and CSF1R signaling, and genes essential for differentiation and function. For example, several miRNAs directly and indirectly target the master osteoclast transcription factor, Nfatc1 (e.g. miR-124 and miR-214) and Rho-GTPases, Cdc42 and Rac1 (e.g. miR-29 family). SUMMARY: Validating miRNA expression patterns, targets, and impact in osteoclasts and other skeletal cells is critical for understanding basic bone biology and for fulfilling the therapeutic potential of miRNA-based strategies in the treatment bone diseases.