inducible-and-reversible-dominant-negative-dn-protein-inhibition

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based ...

Creighton University School of Dentistry

The small bioactive lipid lysophosphatidic acid (LPA) plays critical roles in both normal physiology and inflammation in many systems. However, its actions are just beginning to be defined in oral biology and pathophysiology.

A Major Human Oral Lysophosphatidic Acid Species, LPA 18:1, Regulates Novel Genes in Human Gingival Fibroblasts.

The pleiotropic, bioactive lipid lysophosphatidic acid [(LPA), 1-acyl-sn-glycerol-3-phosphate] exerts critical regulatory actions in physiology and pathophysiology in many systems. It is present in normal bodily fluids, and is elevated in pathology (1). In vivo, "LPA" exists as distinct molecular species, each having a single fatty acid of varying chain length and degree of unsaturation covalently attached to the glycerol backbone via an acyl, alkyl, or alkenyl link. These species differ in affinities for the individual LPA receptors [(LPARs), LPA1-6] and coupling to G proteins (2). However, LPA 18:1 has been and continues to be the most commonly utilized species in reported studies. The actions of "LPA" remain poorly defined in oral biology and pathophysiology. Our laboratory has addressed this knowledge gap by studying in vitro the actions of the major human salivary LPA species [18:1, 18:0, and 16:0 (3)] in human oral cells (4-7). This includes gingival fibroblasts (GF), which our flow cytometry data from multiple donors found that they express LPA1-5 (6). We have also reported that these species are ten-fold elevated to pharmacologic levels in the saliva and gingival crevicular fluid obtained from patients with moderate-severe periodontitis (8). As the potential of LPA to regulate transcriptional activity had not been examined in the oral system, this study used whole human genome microarray analysis to test the hypothesis that LPA 18:1-treated human GF would show significant changes in gene transcripts relevant to their biology, wound-healing, and inflammatory responses. LPA 18:1 was found to significantly regulate a large, complex set of genes critical to GF biology in these categories and to periodontal disease. The raw data has been deposited at NCBI's GEO database as record GSE57496. 

Lysophosphatidic acid (LPA) 18:1 transcriptional regulation of primary human gingival fibroblasts.

Germline mutations in Mir96, one of three co-expressed polycistronic miRNA genes (Mir96, Mir182, Mir183), cause hereditary hearing loss in humans and mice. Transgenic FVB/NCrl- Tg(GFAP-Mir183,Mir96,Mir182)MDW1 mice (Tg), which overexpress this neurosensory-specific miRNA cluster in the inner ear, were developed as a model system to identify, in the aggregate, target genes and biologic processes regulated by the miR-183 cluster. Histological assessments demonstrate Tg homozygotes have a modest increase in cochlear inner hair cells (IHCs). Affymetrix mRNA microarray data analysis revealed that downregulated genes in P5 Tg cochlea are statistically enriched for evolutionarily conserved predicted miR-96, miR-182 or miR-183 target sites. ABR and DPOAE tests from 18 days to 3 months of age revealed that Tg homozygotes develop progressive neurosensory hearing loss that correlates with histologic assessments showing massive losses of both IHCs and outer hair cells (OHCs). This mammalian miRNA misexpression model demonstrates a potency and specificity of cochlear homeostasis for one of the dozens of endogenously co-expressed, evolutionally conserved, small non-protein coding miRNA families. It should be a valuable tool to predict and elucidate miRNA-regulated genes and integrated functional gene expression networks that significantly influence neurosensory cell differentiation, maturation and homeostasis.

Article	Year
A Major Human Oral Lysophosphatidic Acid Species, LPA 18:1, Regulates Novel Genes in Human Gingival Fibroblasts. Journal of periodontology\| PubMed ID: 25660500	2015
Lysophosphatidic acid (LPA) 18:1 transcriptional regulation of primary human gingival fibroblasts. Genomics data\| PubMed ID: 26484133	2014
A mouse model of miR-96, miR-182 and miR-183 misexpression implicates miRNAs in cochlear cell fate and homeostasis. Scientific reports\| PubMed ID: 29476110	2018

Michael D. Weston

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