Heme-oxygenase-1 (HO-1), an important enzyme involved in vascular disease, transplantation, and inflammation, catalyzes the degradation of heme into carbon monoxide and biliverdin. It has been reported that overexpression of HO-1 inhibits osteoclastogenesis. However, the effect of HO-1 on osteoblast differentiation is still not clear. We here used adenoviral vector expressing recombinant human HO-1 and HO-1 inducer hemin to study the effects of HO-1 in primary cultured osteoblasts. The results showed that induction of HO-1 inhibited the maturation of osteoblasts including mineralized bone nodule formation, alkaline phosphatase activity and decreased mRNA expression of several differentiation markers such as alkaline phosphatase, osteocalcin, and RUNX2. Furthermore, downstream products of HO-1, bilirubin, carbon monoxide, and iron, are involved in the inhibitory action of HO-1. HO-1 can be induced by H(2)O(2), lipopolysaccharide and inflammatory cytokines such as TNF-alpha and IL-1beta in osteoblasts and also in STZ-induced diabetic mice. In addition, endogenous PPARgamma ligand, 15-deoxy-Delta(12,14)-prostaglandin-J2 (15d-PGJ2) markedly increased both mRNA and protein levels of HO-1 in osteoblasts via PI3K-Akt and MAPK pathways. Blockade of HO activity by ZnPP IX antagonized the inhibitory action on osteocalcin expression by hemin and 15d-PGJ2. Our results indicate that upregulation of HO-1 inhibits the maturation of osteoblasts and HO-1 may be involved in oxidative- or inflammation-induced bone loss.

SummaryPinin (Pnn), a serine/arginine-rich (SR)-related protein, has been shown to play multiple roles within eukaryotic cells including cell-cell adhesion, cell migration, regulation of gene transcription, mRNA export and alternative splicing. In this study, an attempt to generate mice homozygously deficient in Pnn failed because of early embryonic lethality. To evaluate the effects of loss of Pnn expression on cell survival, RNA interference experiments were performed in MCF-7 cells. Depletion of Pnn resulted in cellular apoptosis and nuclear condensation. In addition, nuclear speckles were disrupted, and expression levels of SR proteins were diminished. RT-PCR analysis showed that alternative splicing patterns of SRSF1 as well as of apoptosis-related genes Bcl-x and ICAD were altered, and expression levels of Bim isoforms were modulated in Pnn-depleted cells. Cellular apoptosis induced by Pnn depletion was rescued by overexpression of SRSF1, which also restored generation of Bcl-xL and functionless ICAD. Pnn expression is, therefore, essential for survival of mouse embryos and the breast carcinoma cell line MCF-7. Moreover, Pnn depletion, modulated by SRSF1, determines cellular apoptosis through activation of the expression of pro-apoptotic Bcl-xS transcripts.

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