Aging is a risk factor for progressive fibrotic disorders involving diverse organ systems, including the lung. Idiopathic pulmonary fibrosis, an age-associated degenerative lung disorder, is characterized by persistence of apoptosis-resistant myofibroblasts. Here we demonstrate that sirtuin 3 (SIRT3), a mitochondrial deacetylase, is downregulated in the lungs of humans with idiopathic pulmonary fibrosis and in mice subjected to lung injury. Overexpression of Sirt3 cDNA via airway delivery restored the capacity for fibrosis resolution in aged mice, in association with activation of the forkhead box transcription factor FoxO3a in fibroblasts, upregulation of pro-apoptotic members of the Bcl2 family and recovery of apoptosis susceptibility. While transforming growth factor-β1 reduced levels of SIRT3 and FOXO3A in lung fibroblasts, cell non-autonomous effects involving macrophage-secreted products were necessary for SIRT3-mediated activation of FOXO3A. Together, these findings reveal a novel role of SIRT3 in pro-resolution macrophage functions that restore susceptibility to apoptosis in fibroblasts via a FOXO3A-dependent mechanism.

Edited by Joel GottesfeldMicroRNAs (miRs) and Hox transcription factors have decisive roles in postnatal bone formation and homeostasis. In silico analysis identified extensive interaction between HOXA cluster mRNA and microRNAs from the miR-23a cluster. However, Hox regulation by the miR-23a cluster during osteoblast differentiation remains undefined. We examined this regulation in preosteoblasts and in a novel miR-23a cluster knockdown mouse model. Overexpression and knockdown of the miR-23a cluster in preosteoblasts decreased and increased, respectively, the expression of the proteins HOXA5, HOXA10, and HOXA11; these proteins' mRNAs exhibited significant binding with the miR-23a cluster miRNAs, and miRNA 3-UTR reporter assays confirmed repression. Importantly, during periods correlating with development and differentiation of bone cells, we found an inverse pattern of expression between HoxA factors and members of the miR-23a cluster. HOXA5 and HOXA11 bound to bone-specific promoters, physically interacted with transcription factor RUNX2, and regulated bone-specific genes. Depletion of HOXA5 or HOXA11 in preosteoblasts also decreased cellular differentiation. Additionally, stable overexpression of the miR-23a cluster in osteoblasts decreased the recruitment of HOXA5 and HOXA11 to osteoblast gene promoters, significantly inhibiting histone H3 acetylation. Heterozygous miR-23a cluster knockdown female mice (miR-23a Cl WT/ZIP ) had significantly increased trabecular bone mass when compared with WT mice. Furthermore, miR-23a cluster knockdown in calvarial osteoblasts of these mice increased the recruitment of HOXA5 and HOXA11, with a substantial enrichment of promoter histone H3 acetylation. Taken together, these findings demonstrate that the miR-23a cluster is required for maintaining stagespecific HoxA factor expression during osteogenesis.Mammalian homeobox (Hox) 3 developmental transcription factors have diverse roles in bone development and postnatal bone formation (1-3). These roles include skeletal element formation (4), anterior-posterior homeotic development (4 -10), and limb and axial skeleton formation (11)(12)(13)(14)(15). In fact, cooperation among Hox genes is critical in skeletal development (2, 13, 16 -18). Moreover, Leucht et al. (19) reported that the Hox gene expression status influences the process of adult bone regeneration. A high-throughput ChIP-sequencing study revealed that HOXD13 binds numerous genes that act in key pathways required for early limb and skeletal patterning (20). Furthermore, Wan and Cao (21) reported that a SMAD-HOX association is required to decipher the mechanism of bone morphogenetic protein signaling in osteoblast growth and differentiation. In previous studies, we demonstrated that selective recruitment of HOX transcription factors to bone-specific chromatin at specific stages of osteoblast maturation mediates gene activation (1,(22)(23)(24). Hence, multiple levels of transcriptional and epigenetic regulation by HOX proteins must be examined to define the complete m...

Multicellular organisms maintain structure and function of tissues/organs through emergent, self-organizing behavior. In this report, we demonstrate a critical role for lung mesenchymal stromal cell (L-MSC) aging in determining the capacity to form three-dimensional organoids or ‘alveolospheres’ with type 2 alveolar epithelial cells (AEC2s). In contrast to L-MSCs from aged mice, young L-MSCs support the efficient formation of alveolospheres when co-cultured with young or aged AEC2s. Aged L-MSCs demonstrated features of cellular senescence, altered bioenergetics, and a senescence-associated secretory profile (SASP). The reactive oxygen species generating enzyme, NADPH oxidase 4 (Nox4), was highly activated in aged L-MSCs and Nox4 downregulation was sufficient to, at least partially, reverse this age-related energy deficit, while restoring the self-organizing capacity of alveolospheres. Together, these data indicate a critical role for cellular bioenergetics and redox homeostasis in an organoid model of self-organization and support the concept of thermodynamic entropy in aging biology.

Fibrosis involving the lung may occur in many settings, including in association with known environmental agents, connective tissue diseases, and exposure to drugs or radiation therapy. The most common form is referred to as ‘idiopathic’ since a causal agent or specific association has not been determined; the strongest risk factor for idiopathic pulmonary fibrosis is aging. Emerging studies indicate that targeting certain components of aging biology may be effective in mitigating age-associated fibrosis. While transforming growth factor-β1 (TGF-β1) is a central mediator of fibrosis in almost all contexts, and across multiple organs, it is not feasible to target this canonical pathway at the ligand–receptor level due to the pleiotropic nature of its actions; importantly, its homeostatic roles as a tumor-suppressor and immune-modulator make this an imprudent strategy. However, defining targets downstream of its receptor(s) that mediate fibrogenesis, while relatively dispenable for tumor- and immune-suppressive functions may aid in developing safer and more effective therapies. In this review, we explore molecular targets that, although TGF-β1 induced/activated, may be relatively more selective in mediating tissue fibrosis. Additionally, we explore epigenetic mechanisms with global effects on the fibrogenic process, as well as metabolic pathways that regulate aging and fibrosis.

Purpose of Review-Precise and temporal expression of Runx2 and its regulatory transcriptional network is a key determinant for the intricate cellular and developmental processes in adult bone tissue formation. This review analyzes how microRNA functions to regulate this network, and how dysregulation results in bone disorders. Recent Findings-Similar to other biologic processes, microRNA (miRNA/miR) regulation is undeniably indispensable to bone synthesis and maintenance. There exists a miRNA-RUNX2 network where RUNX2 regulates the transcription of miRs, or is post transcriptionally regulated by a class of miRs, forming a variety of miR-RUNX2 regulatory pathways which regulate osteogenesis. Summary-The current review provides insights to understand transcriptional-post transcriptional regulatory network governed by Runx2 and osteogenic miRs, and is based largely from in vitro and in vivo studies. When taken together, this article discusses a new regulatory layer of bone tissue specific gene expression by RUNX2 influenced via miRNA.

Chromatin remodeling, specifically the tissue-specific regulation in mineralized tissues, is an understudied avenue of gene regulation. Here we show that Baf45a and Baf45d, two Baf45 homologs belong to ATPase-dependent SWI/SNF chromatin remodeling complex, preferentially expressed in osteoblasts and odontoblasts compared to Baf45b and Baf45c. Recently, biochemical studies revealed that BAF45A associates with Polybromo-associated BAF (PBAF) complex. However, the BAF45D subunit belongs to the polymorphic canonical BRG1-associated factor (cBAF) complex. Protein profiles of osteoblast and odontoblast differentiation uncovered a significant increase of BAF45A and PBAF subunits during early osteoblast and odontoblast maturation. Chromatin immunoprecipitation sequencing (ChIP-seq) during the bone marrow stromal cells (BMSCs) differentiation showed higher histone H3K9 and H3K27 acetylation modifications in the promoter of Baf45a and Baf45d and increased binding of bone and tooth specific transcription factor RUNX2. Overexpression of Baf45a in osteoblasts activates genes essential for the progression of osteoblast maturation and mineralization. Furthermore, shRNA-mediated knockdown of Baf45a in odontoblasts leads to markedly altered genes responsible for the proliferation, apoptosis, DNA repair, and modest decrease in dentinogenic marker gene expression. Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) assay in Baf45a knockout osteoblasts revealed a noticeable reduction in chromatin accessibility of osteoblast and odontoblast specific genes, along with transcription factor Atf4 and Klf4. Craniofacial mesenchyme-specific loss of Baf45a modestly reduced the mineralization of the tooth and mandibular bone. These findings indicated that BAF45A-dependent mineralized tissue-specific chromatin remodeling through PBAF-RUNX2 crosstalk results in transcriptional activation is critical for early differentiation and matrix maturation of mineralized tissues.

Multicellular organisms maintain structure and function of tissues/organs through emergent, self-organizing behavior. In this report, we demonstrate a critical role for lung mesenchymal stromal cell (L-MSC) aging in determining the capacity to form 3-dimentional organoids or alveolospheres with type 2 alveolar epithelial cells (AEC2s). In contrast to L-MSCs from aged mice, young L-MSCs support the efficient formation of alveolospheres when co-cultured with young or aged AEC2s. Aged L-MSCs demonstrated features of cellular senescence, altered bioenergetics, and a senescence-associated secretory profile (SASP). The reactive oxygen species generating enzyme, NADPH oxidase 4 (Nox4), was highly activated in aged L-MSCs and Nox4 downregulation was sufficient to, at least partially, reverse this age-related energy deficit, while restoring the self-organizing capacity of alveolospheres. Together, these data indicate a critical role for cellular bioenergetics and redox homeostasis in an organoid model of self-organization, and supports the concept of thermodynamic entropy in aging biology.

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