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Prostacyclin Synthase Promoter Regulation and Familial Pulmonary Arterial Hypertension^*

^* From the Divisions of Pulmonary Sciences and Critical Care Medicine (Drs. Nana-Sinkam, Stearman, Bull, Grady, Choudhery, and Geraci, Mr. Sotto-Santiago, Mr. Oyer, and Mr. Moore) and Nephrology (Dr. Nemenoff), University of Colorado Health Sciences Center, Denver, CO; and Division of Allergy, Pulmonary and Critical Care Medicine (Drs. Lane and Loyd), Vanderbilt University Medical Center, Nashville, TN.

Correspondence to: Patrick Nana-Sinkam, MD, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, 4200 E Ninth Ave, Box C272, Denver, CO 80262; e-mail: patrick.nana-sinkam{at}uchsc.edu

Primary pulmonary hypertension (PPH) is a rare disease of unknown etiology characterized by sustained elevations in pulmonary artery pressure. Familial PPH accounts for approximately 6% of cases of PPH and has an autosomal-dominant mode of inheritance. Studies over the last 15 years have identified that both familial and sporadic cases of PPH have an association with mutations in the bone morphogenic protein receptor (BMPR) type II gene. Prostacyclin (PGI₂) is currently the standard of care for most patients with severe PPH. Prostacyclin synthase (PGIS) catalyzes the production of PGI₂ from prostaglandin H₂ and is a critical regulatory step in PGI₂ production. Decreased or absent expression of PGIS in the pulmonary microvasculature has been reported in patients with PPH, raising the potential for PGIS as a disease modifying gene. We hypothesize that functional mutations in the PGIS promoter affect transcription regulation of PGI₂ production and play a pivotal role in PPH pathogenesis.

Deletion mutants of the PGIS promoter were constructed in a luciferase reporter system and transfected into vascular smooth-muscle cells (VSMCs) to evaluate promoter activity. A 183–base-pair (bp) region immediately upstream of the start codon was identified to contain maximal promoter activity. This 183-bp region contains a previously reported 9-bp variable-number tandem repeat (VNTR), CCGCCAGCC, that corresponds to activating protein-2 and surfactant protein-1 transcription binding sites. We examined the region within the PGIS promoter in a cohort of familial individuals with known BMPR-II mutations, and control pooled genomic DNA. Promoter constructs were cloned into the same luciferase reporter plasmid and sequenced to examine the distribution of VNTRs in these two populations.

Different distributions of VNTRs, ranging from three to seven repeats, were detected in the two populations (control and familial). The four-repeat allele was underrepresented in the familial BMPR-II group, 12%, compared to 37% in the control group. Further, a five-repeat allele was found at a frequency of 7% in the familial BMPR-II cohort and was absent in the control group. Finally, the different VNTR alleles were transfected into VSMCs to evaluate promoter activity, and a correlation between increased VNTR and promoter activity (p < 0.05) was identified; however, the five-repeat alleles exhibited the lowest activity (p < 0.05).

Decreased or absent expression of PGIS has been reported in patients with PPH, raising the potential for PGIS as a disease-modifying gene. In this study, we examined the DNA sequence of a region within the PGIS promoter and found VNTRs that alter transcription activity of the promoter in a luciferase reporter system when transfected into VSMCs. In addition, the distribution of PGIS promoter VNTR alleles was different between a familial cohort of individuals with BMPRII mutations and a control of pooled genomic DNA. Our findings suggest a potential functional role for the promoter polymorphism in the pathogenesis of PPH. Whether VNTRs correlate with enzyme activity and promoter activity in other cell types have yet to be elucidated and will require further study.

	Footnotes

 
Abbreviations: BMPR = bone morphogenic protein receptor; bp = base pair; PGI₂ = prostacyclin; PGIS = prostacyclin synthase; PPH = primary pulmonary hypertension; VSMC = vascular smooth-muscle cell; VNTR = variable-number tandem repeat

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