Volume 114, Issue 3, Pages 550-558 (March 1998) Expression of activin A is increased in cirrhotic and fibrotic rat livers  Motoya Sugiyama, Takafumi Ichida, Tomomi Sato, Toru Ishikawa, Yasunobu Matsuda, Hitoshi Asakura  Gastroenterology  Volume 114, Issue 3, Pages 550-558 (March 1998) DOI: 10.1016/S0016-5085(98)70539-6 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Kinetics of inhibin βA subunit mRNA expression in rat livers with DMN-induced cirrhosis. Representative Northern blotting of inhibin βA subunit mRNA using total RNA (20 μg) isolated from livers of normal control rats (control, n = 16) and rats treated with DMN for 7 (n = 6), 14 (n = 6), and 21 days (n = 6) in duplicate DMN treatments is shown. The levels of inhibin βA mRNA in control rats did not change during the period of the treatment, and a sample obtained from the day 0 controls is shown as an example. Ribosomal RNAs and GAPDH mRNA bands are represented as internal controls. Northern analysis was performed in triplicate for samples obtained from all rats. kb, kilobases. Gastroenterology 1998 114, 550-558DOI: (10.1016/S0016-5085(98)70539-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Changes in inhibin βA subunit mRNA expression in rat livers with porcine serum–induced fibrosis. Representative Northern blotting using total RNA (20 μg) isolated from livers of normal control rats (control, n = 12) and from rats given porcine serum for 4 weeks (28 days, n = 12) and 8 weeks (56 days, n = 12) in duplicate porcine serum treatments is shown. Inhibin βA mRNA expression in control rats did not change during the period of the treatment, and the data obtained on day 0 are shown as an example. Northern analysis was performed in triplicate for samples obtained from all rats. Gastroenterology 1998 114, 550-558DOI: (10.1016/S0016-5085(98)70539-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Immunohistochemical localization of inhibin βA subunit in the livers of normal rats and rats with DMN-induced cirrhosis. (A and E) Silver staining showed marked accumulation of reticulin fibers around the periportal and pericentral areas after 21 days of DMN treatment. (B and C) Very little immunoreactive inhibin βA subunit was detected in normal livers except for the vascular smooth muscle cells (C, arrow). No inhibin βA subunit immunostaining was observed in hepatocytes. (F and G) After DMN treatment for 21 days, markedly enhanced inhibin βA subunit expression was apparent in hepatocytes with the most intense staining in the perilobular regions located adjacent to the fibrotic areas. (D and H) No immunoreactivity was observed when normal and cirrhotic liver sections were incubated with preimmune rabbit IgG (original magnification: A, B, D–F, and H, 25×; and C and G, 100×). Gastroenterology 1998 114, 550-558DOI: (10.1016/S0016-5085(98)70539-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Immunohistochemical localization of the inhibin βA subunit in rat livers with porcine serum–induced fibrosis. (A) Reticulin fibers spread radially between the portal and central veins without obvious hepatocellular injury. (B and C) Strong inhibin βA immunoreactivity was detected in hepatocytes, especially around the fibrous septa. (D) No positive staining was detected in tissue sections incubated with preimmune rabbit IgG (original magnification: A and D, 25×; B, 50×; and C, 100×). Gastroenterology 1998 114, 550-558DOI: (10.1016/S0016-5085(98)70539-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of TGF-β1 on inhibin βA subunit mRNA expression in primary cultured rat hepatocytes. Primary cultured rat hepatocytes were maintained in the absence (Control) or presence of 1, 2, or 5 ng/mL TGF-β1 for 20 hours, after which 20-μg aliquots of total RNAs were subjected to Northern analysis, which was performed in triplicate. Total RNA isolated from a normal rat ovary (Ovary) which expresses high levels of inhibin βA subunit mRNA, was used as a positive control. Ribosomal RNAs stained with ethidium bromide and the GAPDH mRNA level in each sample are shown to demonstrate RNA loading and integrity. kb, kilobases. Gastroenterology 1998 114, 550-558DOI: (10.1016/S0016-5085(98)70539-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of activin A on inhibin βA subunit mRNA expression in primary cultured rat hepatocytes. Primary cultured hepatocytes were incubated in the absence (Control) or presence of 1, 2, or 5 ng/mL activin A for 20 hours. Twenty micrograms of total RNAs from each sample were subjected to Northern blotting in triplicate. Total RNA of rat normal ovary (Ovary) was used as a positive control. kb, kilobases. Gastroenterology 1998 114, 550-558DOI: (10.1016/S0016-5085(98)70539-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Southern blot analysis of reverse-transcription PCR products obtained with specific type I and II activin receptor primers. Lanes 1–4 contain the reverse-transcription PCR products of RNAs isolated from cultured cells. M, ΦX174-HaeIII–digested DNA marker, Lane 1, rat hepatocytes; Lane 2, rat Ito/fat-storing cells; Lane 3, NRK-49F rat kidney fibroblasts; and Lane 4, negative control (no template cDNA). Southern blotting shows the above gels that hybridized with activin type I and II receptor cDNA probe, respectively. The reverse-transcription PCR products obtained with GAPDH-specific primers are also shown in the bottom panel. Gastroenterology 1998 114, 550-558DOI: (10.1016/S0016-5085(98)70539-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Effects of activin A on type 1 collagen α1(I) mRNA expression in rat Ito/fat-storing cells and NRK-49F fibroblasts. (A) Rat Ito/fat-storing cells and NRK-49F cells were incubated for 20 hours with various concentrations of activin A (0, 1, 10, or 50 ng/mL) in the absence or presence of 5 ng/mL TGF-β1 (TGF-β1(+)). Aliquots (1 μg) of total RNA underwent Northern analysis performed in triplicate as described in Materials and Methods. (B) Relative amounts of collagen α1(I) mRNA were quantified using an image analyzer. Data were from RNA samples obtained from three independent experiments and are expressed as the type 1 collagen α1(I) mRNA/GAPDH mRNA ratio. ■, Rat Ito/fat-storing cells; ▨, NRK-49F cells. The relative density in control cells was assigned as one arbitrary unit. Significance from controls: *P < 0.05; **P < 0.01. Values are expressed as means ± SD. Gastroenterology 1998 114, 550-558DOI: (10.1016/S0016-5085(98)70539-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Effects of activin A on type 1 collagen α1(I) mRNA expression in rat Ito/fat-storing cells and NRK-49F fibroblasts. (A) Rat Ito/fat-storing cells and NRK-49F cells were incubated for 20 hours with various concentrations of activin A (0, 1, 10, or 50 ng/mL) in the absence or presence of 5 ng/mL TGF-β1 (TGF-β1(+)). Aliquots (1 μg) of total RNA underwent Northern analysis performed in triplicate as described in Materials and Methods. (B) Relative amounts of collagen α1(I) mRNA were quantified using an image analyzer. Data were from RNA samples obtained from three independent experiments and are expressed as the type 1 collagen α1(I) mRNA/GAPDH mRNA ratio. ■, Rat Ito/fat-storing cells; ▨, NRK-49F cells. The relative density in control cells was assigned as one arbitrary unit. Significance from controls: *P < 0.05; **P < 0.01. Values are expressed as means ± SD. Gastroenterology 1998 114, 550-558DOI: (10.1016/S0016-5085(98)70539-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions