Kimberly R. Cordes

miR-145 and miR-143 regulate smooth muscle cell fate and plasticity

MicroRNAs (miRNAs) are regulators of myriad cellular events, but evidence for a single miRNA that can efficiently differentiate multipotent stem cells into a specific lineage or regulate direct reprogramming of cells into an alternative cell fate has been elusive. Here we show that miR-145 and miR-143 are co-transcribed in multipotent murine cardiac progenitors before becoming localized to smooth muscle cells, including neural crest stem-cell-derived vascular smooth muscle cells. miR-145 and miR-143 were direct transcriptional targets of serum response factor, myocardin and Nkx2-5 (NK2 transcription factor related, locus 5) and were downregulated in injured or atherosclerotic vessels containing proliferating, less differentiated smooth muscle cells. miR-145 was necessary for myocardin-induced reprogramming of adult fibroblasts into smooth muscle cells and sufficient to induce differentiation of multipotent neural crest stem cells into vascular smooth muscle. Furthermore, miR-145 and miR-143 cooperatively targeted a network of transcription factors, including Klf4 (Kruppel-like factor 4), myocardin and Elk-1 (ELK1, member of ETS oncogene family), to promote differentiation and repress proliferation of smooth muscle cells. These findings demonstrate that miR-145 can direct the smooth muscle fate and that miR-145 and miR-143 function to regulate the quiescent versus proliferative phenotype of smooth muscle cells.

Kidney-specific inactivation of the KIF3A subunit of kinesin-II inhibits renal ciliogenesis and produces polycystic kidney disease

Polycystic kidney disease (PKD) is the most common genetic cause of renal failure in humans. Several proteins that are encoded by genes associated with PKD have recently been identified in primary cilia in renal tubular epithelia. These findings have suggested that abnormalities in cilia formation and function may play a role in the pathogenesis of PKD. To directly determine whether cilia are essential to maintain tubular integrity, we conditionally inactivated KIF3A, a subunit of kinesin-II that is essential for cilia formation, in renal epithelia. Constitutive inactivation of KIF3A produces abnormalities of left–right axis determination and embryonic lethality. Here we show that tissue-specific inactivation of KIF3A in renal tubular epithelial cells results in viable offspring with normal-appearing kidneys at birth. Cysts begin to develop in the kidney at postnatal day 5 and cause renal failure by postnatal day 21. The cyst epithelial cells lack primary cilia and exhibit increased proliferation and apoptosis, apical mislocalization of the epidermal growth factor receptor, increased expression of β-catenin and c-Myc, and inhibition of p21CIP1. These results demonstrate that the absence of renal cilia produces both the clinical and cell biological findings associated with PKD. Most generally, the phenotype of Kif3a mutant mice suggests a role for primary cilia in the maintenance of lumen-forming epithelial differentiation.

Hematopoietic Stem Cells Contribute to the Regeneration of Renal Tubules after Renal Ischemia-Reperfusion Injury in Mice

Ischemia-reperfusion injury (I/R injury) is a common cause of acute renal failure. Recovery from I/R injury requires renal tubular regeneration. Hematopoietic stem cells (HSC) have been shown to be capable of differentiating into hepatocytes, cardiac myocytes, gastrointestinal epithelial cells, and vascular endothelial cells during tissue repair. The current study tested the hypothesis that murine HSC can contribute to the regeneration of renal tubular epithelial cells after I/R injury. HSC isolated from male Rosa26 mice that express beta-galactosidase constitutively were transplanted into female nontransgenic mice after unilateral renal I/R injury. Four weeks after HSC transplantation, beta-galactosidase-positive cells were detected in renal tubules of the recipients by X-Gal staining. PCR analysis of the male-specific Sry gene and Y chromosome fluorescence in situ hybridization confirmed the presence of male-derived cells in the kidneys of female recipients. Antibody co-staining showed that beta-galactosidase was primarily expressed in renal proximal tubules. This is the first report to show that HSC can differentiate into renal tubular cells after I/R injury. Because of their availability, HSC may be useful for cell replacement therapy of acute renal failure.

MicroRNA Regulation of Cardiovascular Development

The transcriptional regulation of cardiovascular development requires precise spatiotemporal control of gene expression, and heterozygous mutations of transcription factors have frequently been implicated in human cardiovascular malformations. A novel mechanism involving posttranscriptional regulation by small, noncoding microRNAs (miRNAs) has emerged as a central regulator of many cardiogenic processes. We are beginning to understand the functions that miRNAs play during essential biological processes, such as cell proliferation, differentiation, apoptosis, stress response, and tumorigenesis. The identification of miRNAs expressed in specific cardiac and vascular cell types has led to the discovery of important regulatory roles for these small RNAs during cardiomyocyte differentiation, cell cycle, conduction, vessel formation, and during stages of cardiac hypertrophy in the adult. Here, we overview the recent findings on miRNA regulation in cardiovascular development and report the latest advances in understanding their function by unveiling their mRNA targets. Further analysis of miRNA function during cardiovascular development will allow us to determine the potential for novel miRNA-based therapeutic strategies.

microRNA-138 modulates cardiac patterning during embryonic development.

Organ patterning during embryonic development requires precise temporal and spatial regulation of protein activity. microRNAs (miRNAs), small noncoding RNAs that typically inhibit protein expression, are broadly important for proper development, but their individual functions during organogenesis are largely unknown. We report that miR-138 is expressed in specific domains in the zebrafish heart and is required to establish appropriate chamber-specific gene expression patterns. Disruption of miR-138 function led to ventricular expansion of gene expression normally restricted to the atrio-ventricular valve region and, ultimately, to disrupted ventricular cardiomyocyte morphology and cardiac function. Temporal-specific knockdown of miR-138 by antagomiRs showed miR-138 function was required during a discrete developmental window, 24–34 h post-fertilization (hpf). miR-138 functioned partially by repressing the retinoic acid synthesis enzyme, aldehyde dehydrogenase-1a2, in the ventricle. This activity was complemented by miR-138-mediated ventricular repression of the gene encoding versican (cspg2), which was positively regulated by retinoic-acid signaling. Our findings demonstrate that miR-138 helps establish discrete domains of gene expression during cardiac morphogenesis by targeting multiple members of a common pathway, and also establish the use of antagomiRs in fish for temporal knockdown of miRNA function.

The neural crest-enriched microRNA miR-452 regulates epithelial-mesenchymal signaling in the first pharyngeal arch

Neural crest cells (NCCs) are a subset of multipotent, migratory stem cells that populate a large number of tissues during development and are important for craniofacial and cardiac morphogenesis. Although microRNAs (miRNAs) have emerged as important regulators of development and disease, little is known about their role in NCC development. Here, we show that loss of miRNA biogenesis by NCC-specific disruption of murine Dicer results in embryos lacking craniofacial cartilaginous structures, cardiac outflow tract septation and thymic and dorsal root ganglia development. Dicer mutant embryos had reduced expression of Dlx2, a transcriptional regulator of pharyngeal arch development, in the first pharyngeal arch (PA1). miR-452 was enriched in NCCs, was sufficient to rescue Dlx2 expression in Dicer mutant pharyngeal arches, and regulated non-cell-autonomous signaling involving Wnt5a, Shh and Fgf8 that converged on Dlx2 regulation in PA1. Correspondingly, knockdown of miR-452 in vivo decreased Dlx2 expression in the mandibular component of PA1, leading to craniofacial defects. These results suggest that post-transcriptional regulation by miRNAs is required for differentiation of NCC-derived tissues and that miR-452 is involved in epithelial-mesenchymal signaling in the pharyngeal arch.

MicroRNAs in Cardiac Development

The transcriptional regulation of cardiovascular development requires precise spatiotemporal control of gene expression, and heterozygous mutations of transcription factors have frequently been implicated in human cardiovascular malformations. A novel mechanism involving post-transcriptional regulation by small, noncoding microRNAs (miRNAs) has emerged as a central regulator of many cardiogenic processes. We are beginning to understand the functions that miRNAs play during essential biologic processes, such as cell proliferation, differentiation, apoptosis, stress response, and tumorigenesis. The identification of miRNAs expressed in specific cardiac and vascular cell types has led to the discovery of important regulatory roles for these small RNAs during cardiomyocyte differentiation, cell cycle, conduction, and vessel formation. Here, we overview the recent findings on miRNA regulation in cardiovascular development. Further analysis of miRNA function during cardiovascular development will allow us to determine the potential for novel miRNA-based therapeutic strategies.

Developmental Changes in Proximal Tubule Tight Junction Proteins

We demonstrated previously that neonatal proximal tubules have a lower passive paracellular permeability to chloride ions and higher resistance than that of adult proximal tubules. In addition, administration of thyroid hormone to neonates, before the normal maturational increase in serum thyroid hormone levels, prematurely accelerates the developmental increase in chloride permeability to adult levels. To test the hypothesis that there is a maturational change in tight junction proteins and that thyroid hormone mediates these changes, we examined the two known tight junction proteins present in proximal tubules, occludin and claudin 2. Using immunoblot and immunohistochemistry, we demonstrated that claudin 2 has a 4-fold greater abundance in neonatal proximal tubules than in adult tubules. Occludin, however, has a 4-fold greater expression in adult tubules than in neonatal tubules. Administration of thyroid hormone to neonates did not affect claudin 2 expression, occludin expression, or the transepithelial resistance in rat proximal tubule cells in vitro. In conclusion, there are postnatal maturational changes in tight junction proteins. The factors that cause these maturational changes are unknown but unlikely to be due solely to the maturational increase in thyroid hormone.

MicroRNA Regulation of Cardiac Development and Disease

Publisher Summary This chapter reviews the basic mechanisms by which miRNAs function, with a focus on the role of miRNAs during development and maintenance of the heart and vessels. It also discusses the function and regulation of specific miRNAs and their mRNA and the mechanisms that regulate gene expression and that can lead to new and distinct therapeutic targets for heart disease. The miRNAs play fascinating roles in the heart, both pre- and postnatally. Through their ability to post-transcriptionally regulate mRNA levels, and thus manage protein dosage, miRNAs provide finer regulation within the complex molecular networks that regulate cardiogenesis. The importance of this fine regulation is highlighted by the recognition that most known genetic causes of heart malformations in humans result from haploinsufficiency or heterozygous point mutations. The field of miRNA biology is growing rapidly, and new tools and mechanisms are becoming available. With further characterization, elucidating the function of cardiac-enriched miRNAs may provide us with new diagnostic, prognostic, and therapeutic targets for many forms of cardiovascular disease.