Nitric oxide (NO) regulates lung development through incompletely understood mechanisms. NO controls pulmonary vascular smooth muscle cell (SMC) differentiation largely through stimulating soluble guanylate cyclase (sGC) to produce cGMP and increase cGMP-mediated signaling. To examine the role of sGC in regulating pulmonary development, we tested whether decreased sGC activity reduces alveolarization in the normal and injured newborn lung. For these studies, mouse pups with gene-targeted sGC-α1 subunit truncation were used because we determined that they have decreased pulmonary sGC enzyme activity. sGC-α1 knockout (KO) mouse pups were observed to have decreased numbers of small airway structures and lung volume compared with wild-type (WT) mice although lung septation and body weights were not different. However, following mild lung injury caused by breathing 70% O2, the sGC-α1 KO mouse pups had pronounced inhibition of alveolarization, as evidenced by an increase in airway mean linear intercept, reduction in terminal airway units, and decrease in lung septation and alveolar openings, as well as reduced somatic growth. Because cGMP regulates SMC phenotype, we also tested whether decreased sGC activity reduces lung myofibroblast differentiation. Cellular markers revealed that vascular SMC differentiation decreased, whereas myofibroblast activation increased in the hyperoxic sGC-α1 KO pup lung. These results indicate that lung development, particularly during hyperoxic injury, is impaired in mouse pups with diminished sGC activity. These studies support the investigation of sGC-targeting agents as therapies directed at improving development in the newborn lung exposed to injury.

cGMP-dependent protein kinase I (PKGI) is an important effector of cGMP signaling that regulates vascular smooth muscle cell (SMC) phenotype and proliferation. PKGI has been detected in the perinuclear region of cells, and recent data indicate that proprotein convertases (PCs) typically resident in the Golgi apparatus (GA) can stimulate PKGI proteolysis and generate a kinase fragment that localizes to the nucleus and regulates gene expression. However, the role of the endomembrane system in PKGI compartmentation and processing is unknown. Here, we demonstrate that PKGI colocalizes with endoplasmic reticulum (ER), ER-Golgi intermediate compartment, GA cisterna, and trans-Golgi network proteins in pulmonary artery SMC and cell lines. Moreover, PKGI localizes with furin, a trans-Golgi network-resident PC known to cleave PKGI. ER protein transport influences PKGI localization because overexpression of a constitutively inactive Sar1 transgene caused PKGI retention in the ER. Additionally, PKGI appears to reside within the GA because PKGI immunoreactivity was determined to be resistant to cytosolic proteinase K treatment in live cells. The GA appears to play a role in PKGI proteolysis because overexpression of inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, not only tethered heterologous PKGI-β to the ER and decreased its localization to the GA, but also diminished PKGI proteolysis and nuclear translocation. Also, inhibiting intra-GA protein transport with monensin was observed to decrease PKGI cleavage. These studies detail a role for the endomembrane system in regulating PKGI compartmentation and proteolysis. Moreover, they support the investigation of mechanisms regulating PKGI-dependent nuclear cGMP signaling in the pulmonary vasculature with Golgi dysfunction.

cGMP regulates vascular tone and structure through stimulating PKGI. Although encoded by a single gene product, two PKGI isoforms, PKGIα and PKGIβ, form through alternate mRNA splicing. They differ in their NH2‐terminal dimerization domains. Proteolysis of the PKGI isoforms and the nuclear localization of their conserved COOH‐terminal kinase fragment (PKGIγ) play a critical role in regulating SMC gene expression and differentiation. Although PKGI has been detected in the perinuclear region of SMC, and proprotein convertases (PCs) that are resident in the Golgi apparatus (GA) have been found to stimulate PKGI proteolysis, the role of the GA in PKGI processing is not understood. Using pulmonary artery SMC and rat fetal lung fibroblast (RFL‐6) cells, which express endogenous PKGI, we determined that PKGI immunoreactivity co‐localizes with index proteins residing in the endoplasmic reticulum (ER), ER‐Golgi intermediate complex, GA cisterna, and the trans‐Golgi network, including furin a PKGI‐cleaving PC. Moreover, over‐expression of Sar1 [T39N], a GDP‐bound trans‐dominant Sar1 mutant that inhibits ER exit site budding, was found to inhibit PKGI localization to GA cisterna. Also, IP3R‐associated cGMP kinase substrate (IRAG) was found not only to tether overexpressed PKGIβ to the ER and decrease its GA localization, but also to decrease PKGI proteolysis and PKGIγ nuclear translocation. Monensin, which inhibits intra‐GA protein transport, was also found to decrease PKGI cleavage. Together these studies detail for the first time a role for ER‐GA transport in PKGI post‐translational modifications that regulate nuclear signaling. NIH R01HL096779 supported this work.