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First published online February 18, 2004
doi: 10.1242/10.1242/dev.00999

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HtrA1 serine protease inhibits signaling mediated by Tgfß family proteins

¹ Division of Gene Function in Animals, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
² Division of Metabolic Regulation of Animal Cells, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan

View larger version (16K): [in a new window]   Fig. 5. Binding to and inhibition of Bmp4 signaling by HtrA1 mutants. Schematic presentation of each HtrA1 mutant is shown on the left. Numbers indicate the amino acid residues. The table on the right summarizes the activities of HtrA1 mutants to bind and inhibit Bmp4. The binding activity was designated as `+' when higher than 3% but less than 10% of input HtrA1 mutant protein was recovered by the GST pull-down assay, and `-' when the recovery was less than 1%. Inhibitory activity of the wild-type HtrA1 was defined as 100% for each amount of vector DNA (50 or 100 ng) added to the well. SS, signal sequence.

View larger version (90K): [in a new window]   Fig. 2. HtrA1 expression during the development of skeletal elements. (A) Whole-mount in situ hybridization of the 13.5 dpc forelimb. (B) Section of A. (C) In situ hybridization of a transverse section of the 14.5 dpc limb. (D) In situ hybridization of a 14.5 dpc rib section. (E) Immunostaining of a 15.5 dpc rib section. The localization of HtrA1 protein is in good agreement with that of mRNA shown in D. (F) In situ hybridization of the 17.5 dpc hind limb. Inset shows a magnified view of the boxed area. Low expression can be seen in a thin layer of the articular surface (arrowhead in inset). (G,H,I) Immunostaining of adult tibia. HtrA1 is detected in the bone matrix of the diaphysis and epiphysis. ac, articular cartilage; bm, bone marrow; c, cartilage nodule; ca, calcaneus; cu, cuboid; et, extensor tendon primordium; gp, growth plate; hc, hypertrophic cartilage; j, joint developing region; l, lateral cuneiform; oc, ossification center; pc, proliferating cartilage; pch, perichondrium; po, periosteum; t, tendon; III, digit III; 3m, 3rd metatarsal.

View larger version (154K): [in a new window]   Fig. 1. Expression pattern of HtrA1. (A) Whole-mount in situ hybridization of 12.5 dpc embryo. Embryos were hybridized with a sense probe (left), or with an antisense probe (right). (B) In situ hybridization of a sagittal section of 12.5 dpc mouse embryo. HtrA1 expression (blue signals) is seen in the vertebral column, hind limb, brain, gonad, heart, lung and abdominal skin. (C) In situ hybridization of a sagittal section of 14.5 dpc embryo. In addition to the sites mentioned in B, HtrA1 expression is seen in the intervertebral disc, tracheal wall, cardiac outflow tracts and meninges. In the hind limb, HtrA1 is expressed in the primordia of the extensor tendons. (D) Magnified view of B showing the vertebral column and ribs. (E) Magnified view of C showing the brain area near the fourth ventricle. Note that the meninges also expressed HtrA1. (F) Immunostaining of a transverse section through the heart at 11.5 dpc. HtrA1 protein (brown) is detected in the AV cushion and outflow tract. (G) Magnified view of B showing the heart. HtrA1 is expressed in the AV endocardial cushion. (H) Magnified view of B showing the gonad. The Wolffian duct does not express HtrA1 but the mesonephric tubules do. (I) Magnified view of B showing the lung. (J) In situ hybridization of a section of 14.5 dpc maxillary skin. (K,L) Immunostaining of the skin. The eyelid of an 18.5 dpc embryo (K) and the skin from the back of a 9-day old pup (L) are shown. HtrA1 protein is detected in the basal layer of epidermis (arrow), in the epithelial component of the developing whisker (arrowhead). HtrA1 protein is also detected in the outer root sheets of whiskers of the 9-day pup. a, cardiac atria; ao, aorta; as, abdominal skin; av, atrioventricular cushion; b, bronchicles; c, condensing cartilage; cp, choroid plexus; d, dermis; e, epidermis; et, primordium of extensor tendon; g, gonad primordium; h, heart; hb, hind brain; hl, hind limb; hs, hair shaft; id, intervertebral disc; in, intestine; irs, inner root sheath; li, liver; lu, lung; M, Müllerian duct; m, meninges; mc, metacephalon; me, mesonephric tubules; nt, neural tube; ot, outflow tract; ors, outer root sheath; r, rib; t, tendon/ligament; tc, telencephalon; tr, trachea; v, cardiac ventricle; ve, vertebral column; W, Wolffian duct; wf, whisker follicle; 4v, 4th ventricle.

View larger version (29K): [in a new window]   Fig. 3. Binding of HtrA1 to various Tgfß family proteins. (A) Binding of HtrA1 and follistatin to Tgfß family proteins. GST pull-down assay was carried out using GST-Tgfß fusion proteins and either HtrA1 (b) or myc-tagged follistatin (a) in the presence 1.0 M NaCl. Bound HtrA1 or follistatin was detected by western blotting. The righthand lane in each panel (asterisk) was loaded with 3% input HtrA1 or follistatin. Lower panels show the recovered GST-Tgfß fusion proteins stained with Coomassie blue. GST alone did not bind to HtrA1 (left-hand lanes). (B) Binding of mutant HtrA1 proteins to Bmp4 or Gdf5. Results of a GST pull-down assay with FS (a), FS/PDZ (b), linker/SP/PDZ (c) and S328A (d) are shown. One or two lanes on the right were loaded with aliquots of input HtrA1 mutants; 10% (**) or 1% (*) of input in a and c, 20% (**) or 2%(*) in b, and 15% (*) of input in d. Arrowheads indicate positions of HtrA1 mutant proteins. (C) Solid phase binding assay of HtrA1. ELISA plate wells coated with GST or GST-Bmp4 were incubated with 100 µl of a solution containing myc-tagged HtrA1 protein (0.01-0.3 µg/ml). The binding of HtrA1 to GST (white triangle) or GST-Bmp4 (black square) was quantitated by colorimetric assay using anti-myc antibody.

View larger version (26K): [in a new window]   Fig. 4. Inhibition of Bmp4 or Tgfß1 signaling by HtrA1. (A) Inhibition of Bmp4 signaling by HtrA1. C2C12 cells were transfected in 24-well plates with 250 ng/well pGL3-Id985WT and 50 ng/well expression vectors for Smad1, Smad4 and Bmp4. The expression vectors for HtrA1 (pcDNA3-HtrA1) or noggin (pEF-Bosnoggin) were co-transfected as indicated below the figure. (B) Inhibition of Tgfß1 signaling by HtrA1. C2C12 cells were transfected in 24-well plates with 250 ng/well pGL3ti-(SBE)4 and 50 ng/well Smad1 expression vector. Recombinant human Tgfß1 protein (10 ng/ml) was added 24 hours after transfection. The expression vectors for HtrA1 and noggin were co-transfected as indicated below the figure. (C) Inhibition of Bmp4 signaling by various HtrA1 mutants. The expression vectors for FS, FS/PDZ, linker/FS/PDZ or S328A were co-transfected as in A. Insets show results of western blots, showing the expression levels of wild-type HtrA1 and its mutants, other than FS/PDZ, at 50, 100 and 200 ng DNA/well. Data are the average of three experiments. Standard deviations are shown as a line on each bar. (D) HtrA1 did not inhibit the signal that originated from the constitutively active Bmp receptor. C2C12 cells were transfected as described in A, except that the expression vector of Bmp4 was replaced with pEF-Bos-caBMPR-IB (3 ng/well). The amount of pEF-Bos-caBMPR-IB was adjusted so that it gave similar transcriptional activation to that of the Bmp4 vector (50 ng/well; see the right half of the panel).

View larger version (72K): [in a new window]   Fig. 6. HtrA1 antagonizes Bmp signaling in vivo. Cultured 293T cells expressing HtrA1 (A-I) or noggin (J-R) were injected near the right eyes of stage 9-10 chick embryos, or the HtrA1 expression vector was electroporated into the right optic cups of stage 10-11 chick embryos (S,T,U). After incubation until stage 23-24 (A-R) or stage 17-18 (S,T,U), embryos were harvested. (A,J) Localization of injected cells as monitored by GFP fluorescence. Pictures of the eyes were taken at a constant magnification and image-analyzed for size. The lens or retina was judged to be small when the area of the lens or the pigmented retina in the picture was less than 70% of the untreated side. (B,K) Eyes in un-injected sides. (C,L) Eyes injected with HtrA1- or noggin-expressing cells. (D,M) Frontal sections of control eyes. (E,N) Frontal sections of injected eyes. (F,H,O,Q) Magnified views of D and M. (G,I,P,R) Magnified views of E and N, showing abnormalities of the lens and ventral retina caused by HtrA1 and noggin, respectively. Note that the retinal pigment epithelium (regions between arrowheads in D and M) was replaced by a neuroepithelium-like tissue (regions between arrowheads in E and N). Vertical lines in F, G, O and P indicate the thickness of the primary lens fibers. (S) Retina showing expression of co-electroporated GFP. (T) Vax expression in the untreated eye. (U) Upregulated Vax expression in the retina caused by overexpression of HtrA1. Eyes tended to be smaller after electroporation with the HtrA1 expression vector. Upregulation of Vax expression was seen in eyes of normal or slightly small size.