Toshiko Fujita

Purification of senescence marker protein-30 (SMP30) and its androgen-independent decrease with age in the rat liver.

Age-associated changes in the soluble proteins from rat liver were examined by a newly developed two-dimensional cellulose acetate membrane electrophoresis (2D-CAME). We detected and isolated a novel rat liver protein, the amounts of which decreased androgen-independently with aging. We designated this protein, whose molecular mass was 30 kDa and pI value was 4.9, as senescence marker protein-30 (SMP30). The expression of SMP30 was not modified by castration or treatment with testosterone propionate after castration. Age-associated decrease of SMP30 level was also recognized in aged female. We noted another protein with a pI value of 7.3 that decreased with aging. Its expression seemed to be androgen-dependent and it was markedly expressed only in young and adult male rat livers. We then purified SMP30 and prepared an anti-serum to SMP30. Immunohistochemical analysis showed that the localization of SMP30 was restricted to liver and kidney among numerous organs tested. Although the amount of SMP30 in the liver was relatively large and the tissue distribution was characteristic, no known protein corresponds to this protein.

Senescence marker protein-30 knockout mouse liver is highly susceptible to tumor necrosis factor-α- and fas-mediated apoptosis

Senescence marker protein-30 (SMP30) is a calcium-binding protein that decreases in an androgen-independent manner with aging. To elucidate the physiological role of this protein, we introduced a null mutation of the SMP30 gene into the germ line of mice. Despite the complete lack of SMP30 (SMP30-/-), these mutant mice were indistinguishable from their wild-type (SMP30+/+) littermates in terms of development and fertilization capability. We then investigated the tissue susceptibility for apoptosis induced by cytokine using primary cultured hepatocytes, because SMP30 could rescue cells from cell death caused by calcium influx, using a calcium ionophore as previously described. SMP30-/- hepatocytes were found to be more susceptible to apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) plus actinomycin D (ActD) than SMP30+/+ hepatocytes. In addition, the TNF-alpha/ActD-induced caspase-8 activity in SMP30-/- hepatocytes was twofold greater than that in SMP30+/+ hepatocytes. In contrast, no significant difference was observed in the TNF-alpha/ActD-induced nuclear factor-kappa B activation of SMP30+/+ versus SMP30-/- hepatocytes, indicating that SMP30 is not related to TNF-alpha/ActD-induced nuclear factor-kappa B activation itself. Moreover, deletion of the SMP30 gene enhanced liver injury after treatment in vivo with anti-Fas antibody and the SMP30+/- mice showed intermediate susceptibility to Fas-induced apoptosis. Collectively, these results demonstrate that SMP30 acts to protect cells from apoptosis.

Isolation of cDNA clone encoding human homologue of senescence marker protein-30 (SMP30) and its location on the X chromosome

We have isolated and characterized a cDNA clone encoding human homologue of senescence marker protein-30 (SMP30), a calcium binding protein also called regucalcin (RC). This clone (pHSMP6) has 1356 base pairs (bp) and contains an open reading frame of 897 bp, which encodes 299 amino acids. The estimated molecular weight of the deduced polypeptide is 33,250 and pI is 5.836. The homology of amino acid sequences between human homologue and rat SMP30 is 88.6%. Using pHSMP6 as a probe, the chromosomal location of the human homologue of SMP30 gene was determined. The results of regional mapping using a panel of 11 rodent-human somatic hybrids indicated that the gene is located in the p11.3-q11.2 segment of the X chromosome. This gene thus could be a candidate for one of the X-linked diseases mapped to this regions.

Gene regulation of senescence marker protein-30 (SMP30) : coordinated up-regulation with tissue maturation and gradual down-regulation with aging

Senescence marker protein-30 (SMP30) is a calcium binding protein also called regucalcin. The amounts of SMP30 decrease androgen-independently with aging in the livers of rats. We have studied the expression of SMP30 in livers and kidneys of rats from the embryonic to the senescent stages of life. No transcript was detected in livers or kidneys in day 18 embryos. However, Northern blot analysis showed a marked increase of SMP30 mRNA in livers of neonatal and young rats. The first peak of SMP30 transcript was found in a 5-day-old neonate, in which the amount of mRNA was threefold higher in comparison with 3- to 6.5-month-old adults. The expression of SMP30 protein started to increase from day 7 and rapidly reached a plateau at day 10. The substantial amounts of protein and transcript were maintained in adults up to 3-6.5 months of age. In the kidney, SMP30 mRNA and protein started to increase at day 21 and reached near-maximal levels at day 35. The levels of transcript and protein remained high in adults up to 3 months of age. As the aging process progressed to senescent stages, the levels of transcript and protein decreased significantly in the liver and kidney of aged rats. Therefore, the age-associated decrease of SMP30 in the liver and kidney may be, in a large part, controlled at transcriptional levels. Furthermore, immunohistochemical analysis showed a similar pattern of changes in SMP30 protein expression during neonate, adult and senescent stages in hepatocytes and renal proximal tubular epithelia. The high expression of SMP30 in the tissue-maturing process and adulthood suggests that SMP30 may be required for the maintenance of highly differentiated hepatic and renal functions.

Regulatory effects of senescence marker protein 30 on the proliferation of hepatocytes

Senescence marker protein 30 (SMP 30) is preferentially expressed in the liver. One of its remarkable functions is the protection of cells against various injuries by enhancement of membrane calcium‐pump activity. We analyzed the role of SMP 30 in hepatocyte proliferation. SMP 30 expression was decreased initially, then increased along with hepatic regeneration, after carbon tetrachloride (CCl4) administration. SMP 30 expression was decreased in the necrotic phase and then gradually increased. Its increase was slightly delayed just after the mitotic phase. These results lead us to speculate that mitoses of hepatic cells induce enhanced SMP 30 expression. In contrast, administration of lead nitrate (LN) as a hepatic mitogen induced a more stable increase of SMP 30 expression. To estimate the effect of SMP 30 on cell proliferation, we evaluated hepatic mitosis in wild‐type and SMP 30‐deficient knockout (KO) mice after CCl4 administration. We found an increase in mitotic numbers in hepatocytes of KO mice. This result suggests that SMP 30 has a suppressive effect on cell proliferation. Suppressive activity of SMP 30 cDNA was shown in cultured hepatoblastic cells. Our results suggest that SMP 30 performs a regulatory function in liver regeneration.

Senescence marker protein-30 (SMP30) enhances the calcium efflux from renal tubular epithelial cells

AbstractBackground. Senescence marker protein-30 (SMP30), a calcium binding protein, is preferentially expressed in the renal proximal tubules and hepatocytes and is presumed to play a role in Ca2+ homeostasis. Methods. To explore its physiological functions in the tubular cells, we investigated the effect of SMP30 on Ca2+ efflux via the ATP-dependent plasma membrane calcium pump. LLC-PK1 cells were stably transfected with a cDNA encoding SMP30, and the established transfectants were subjected to ATP responses. Results. Overexpression of SMP30 significantly increased Ca2+ efflux under both basal and ATP-stimulated conditions. Inhibition of calmodulin by trifluoperazine abrogated the enhanced Ca2+ efflux, suggesting that SMP30 activated the calmodulin-dependent Ca2+ pump. It is known that Ca2+ superfluous influx induces cellular injury. Compared with mock-transfected cells, LLC-PK1 cells expressing SMP30 showed resistance to cellular death triggered by Ca2+ superfluous influx. Conclusion. These results suggest the possibility that, in renal tubular cells, endogenous SMP30 participates in Ca2+ efflux via activating the calmodulin-dependent Ca2+ pump and thereby confers resistance of the cells against injury caused by high intracellular Ca2+ concentrations.

Elevated levels of c-jun and c-fos transcripts in the aged rat liver.

Age-associated increases of transcript levels of several genes were studied using aged rat liver. Northern hybridization showed marked increases of c-jun, c-fos, and glutathione S-transferase P (GST-P) in 24-month-old rats. These genes have the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-responsive element called TRE in their structures. Thus, TRE seems to be involved in important age-associated changes of gene expression.

Senescence marker protein-30 (SMP30) induces formation of microvilli and bile canaliculi in Hep G2 cells.

Senescence marker protein-30 (SMP30) is an androgen-independent factor that decreases with aging. To elucidate the physiological functions of SMP30, we transfected human SMP30 cDNA into the human hepatoma cell line, Hep G2. These Hep G2/SMP30 transfectants, which stably expressed large amounts of SMP30, proliferated at a slower rate and synthesized less DNA than mock transfectants (Hep G2/pcDNA3 controls). Thus, enhanced expression of SMP30 retarded the growth of Hep G2/SMP30 cells. Ultrastructural studies by scanning electron microscopy revealed numerous microvilli covering the surfaces of Hep G2/SMP30 cells, whereas few microvilli appeared on control cells. Subsequently, transmission electron microscopy revealed that groups of Hep G2/SMP30 cells exhibited bile canaliculi and possessed specialized adhesion contacts, such as tight junctions and desmosomes, at interplasmic membranes. However, in controls, units of only two cells were seen, and these lacked specialized adhesion junctions. Moesin and ZO-1 are known to be concentrated in microvilli and at tight junctions, respectively. Double-immunostaining was performed to examine whether moesin and ZO-1 were expressed in bile canaliculi with microvilli at the apical regions of Hep G2/SMP30 cells. The intensity of moesin and ZO-1 staining in the contact regions of each cell was markedly higher in Hep G2/SMP30 than in control cells. Moreover, moesin stained more interior areas, which corresponded to the microvilli of bile canaliculi. Clearly, bile canaliculi with microvilli formed at the apical ends of Hep G2/SMP30 cells. These results indicate that SMP30 has an important physiological function as a participant in cell-to-cell interactions and imply that the down-regulation of SMP30 during the aging process contributes to the deterioration of cellular interactivity.

Hepatic and renal expression of senescence marker protein-30 and its biological significance.

A novel rat hepatic protein was detected and isolated, the amount of which is down‐regulated in an androgen‐independent manner with ageing. This protein was designated as senescence marker protein‐30 (SMP30). Senescence marker protein‐30 turned out to be identical to a hepatic calcium‐binding protein called regucalcin (RC). This review gives an overview of SMP30 in its structure, expression and possible physiological function(s). A hypothetical role of SMP30 in ageing and calcium homeostasis is also discussed.