HtrA1, a member of the mammalian HtrA serine protease family, has a highly conserved protease domain followed by a PDZ domain. Because HtrA1 is a secretory protein and has another functional domain with homology to follistatin, we examined whether HtrA1 functions as an antagonist of Tgfβfamily proteins. During embryo development, mouse HtrA1 was expressed in specific areas where signaling by Tgfβ family proteins plays important regulatory roles. The GST-pulldown assay showed that HtrA1 binds to a broad range of Tgfβ family proteins, including Bmp4, Gdf5, Tgfβs and activin. HtrA1 inhibited signaling by Bmp4, Bmp2, and Tgfβ1 in C2C12 cells, presumably by preventing receptor activation. Experiments using a series of deletion mutants indicated that the binding activity of HtrA1 required the protease domain and a small linker region preceding it, and that inhibition of Tgfβ signaling is dependent on the proteolytic activity of HtrA1. Misexpression of HtrA1 near the developing chick eye led to suppression of eye development that was indistinguishable from the effects of noggin. Taken together, these data indicate that HtrA1 protease is a novel inhibitor of Tgfβ family members.

The expression of mouse HtrA1 is developmentally regulated and restricted in embryo tissues which depend largely on TGF‐β signaling for their differentiation. We examined whether mouse HtrA3, another HtrA family member very close to HtrA1, shows similar expression patterns. HtrA3 and ‐1 were expressed mostly in the same embryonic organs but exhibited complementary patterns in various tissues; the lens epithelial cells in day 12.5 embryo expressed HtrA3 whereas the ciliary body and pigment retina expressed HtrA1. In the vertebrae of day 14.5 embryo, HtrA3 was expressed in the tail region, but HtrA1 was predominantly expressed in the thoracic and lumbar regions. Similar to HtrA1, HtrA3 bound to various TGF‐β proteins and inhibited the signaling of BMP‐4, ‐2 and TGF‐β1. HtrA3 did not inhibit signaling originated from a constitutively active BMP receptor, indicating that the inhibition occurred upstream of the cell surface receptor. HtrA3 also showed proteolytic activities indistinguishable from those of HtrA1 toward β‐casein and some extracellular matrix (ECM) proteoglycans. The protease activity was absolutely required for the TGF‐β signal inhibition activity. All these data suggest that HtrA3 and ‐1 have the overlapping biological activities but can function in complementary fashion in certain types of tissues.

Higher-order motor cortices, such as the secondary motor area (M2) in rodents, select future action patterns and transmit them to the primary motor cortex (M1). To better understand motor processing, we characterized "top-down" and "bottom-up" connectivities between M1 and M2 in the rat cortex. Somata of pyramidal cells (PCs) in M2 projecting to M1 were distributed in lower layer 2/3 (L2/3) and upper layer 5 (L5), whereas PCs projecting from M1 to M2 had somata distributed throughout L2/3 and L5. M2 afferents terminated preferentially in upper layer 1 of M1, which also receives indirect basal ganglia output through afferents from the ventral anterior and ventromedial thalamic nuclei. On the other hand, M1 afferents terminated preferentially in L2/3 of M2, a zone receiving indirect cerebellar output through thalamic afferents from the ventrolateral nucleus. While L5 corticopontine (CPn) cells with collaterals to the spinal cord did not participate in corticocortical projections, CPn cells with collaterals to the thalamus contributed preferentially to connections from M2 to M1. L5 callosal projection (commissural) cells participated in connectivity between M1 and M2 bidirectionally. We conclude that the connectivity between M1 and M2 is directionally specialized, involving specific PC subtypes that selectively target lamina receiving distinct thalamocortical inputs.

HtrA1, a member of the mammalian HtrA (high temperature requirement A) serine protease family, has a highly conserved protease domain followed by a PDZ domain. Accumulating evidence has indicated that PDZ domains regulate protease activity of HtrA proteins. We searched for binding partners of the PDZ domain of mouse HtrA1 by yeast two-hybrid screening, and isolated proteins that were recognized by the HtrA1 PDZ domain through their C-terminal ends with a core consensus Phi-X-Phi-[V/L/F/A]-COOH sequence (where Phi is a hydrophobic/non-polar amino acid). C-propeptides of fibrillar collagens were most frequently isolated. Type III procollagen alpha1 C-propeptide, which was used as a model protein, was digested by HtrA1. HtrA1 cleavage of the collagen C-propeptide was enhanced by reductive denaturation of the C-propeptide and partly inhibited by removal of the C-terminal four amino acids from the C-propeptide, suggesting that the substrate recognition was facilitated by the binding of the free C-terminal ends of substrates to the PDZ domain of HtrA1. The synthetic oligopeptide (GM130Pep) that fitted the consensus recognition sequence bound to HtrA1 with a high affinity (K(d)=6.0 nM). GM130Pep stimulated HtrA1 protease activity 3- to 4-fold, but did not efficiently stimulate the activity of an HtrA1 mutant lacking the PDZ domain, supporting the notion that the PDZ domain enhances protease activity upon ligand binding. The peptide derived from Type III collagen alpha1 C-propeptide specifically stimulated protease activity of HtrA1, but did not stimulate nor significantly bind to HtrA2, suggesting that the collagen C-propeptide is a specific physiological regulator of HtrA1.