Degradation of invariant chain (Ii) is a critical step in major histocompatibility complex class II-restricted antigen presentation. Cathepsin L was found to be necessary for Ii degradation in cortical thymic epithelial cells (cTECs), but not in bone marrow (BM)-derived antigen-presenting cells (APCs). Consequently, positive selection of CD4+ T cells was reduced. Because different cysteine proteinases are responsible for specific Ii degradation steps in cTECs and BM-derived APCs, the proteolytic environment in cells mediating positive and negative selection may be distinct. The identification of a protease involved in class II presentation in a tissue-specific manner suggests a potential means of manipulating CD4+ T cell responsiveness in vivo.

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The induction of developmental structures derived from the ectoderm, such as the neural tube or tooth, occurs through neutralization of the inhibitory activity of members of the bone-morphogenetic protein (BMP) family by BMP antagonists. Here we show that, during hair-follicle development, the neural inducer and BMP-neutralizing protein Noggin is expressed in the follicular mesenchyme, that noggin-knockout mice show significant retardation of hair-follicle induction, and that Noggin neutralizes the inhibitory action of BMP-4 and stimulates hair-follicle induction in embryonic skin organ culture. As a crucial mesenchymal signal that stimulates hair-follicle induction, Noggin operates through antagonistic interactions with BMP-4, which result in upregulation of the transcription factor Lef-1 and the cell-adhesion molecule NCAM, as well as through BMP4-independent downregulation of the 75 kD neurotrophin receptor in the developing hair follicle.

Lysosomal cysteine proteinases of the papain family are involved in lysosomal bulk proteolysis, major histocompatibility complex class II mediated antigen presentation, prohormone processing, and extracellular matrix remodeling. Cathepsin L (CTSL) is a ubiquitously expressed major representative of the papain-like family of cysteine proteinases. To investigate CTSL in vivo functions, the gene was inactivated by gene targeting in embryonic stem cells. CTSL-deficient mice develop periodic hair loss and epidermal hyperplasia, acanthosis, and hyperkeratosis. The hair loss is due to alterations of hair follicle morphogenesis and cycling, dilatation of hair follicle canals, and disturbed club hair formation. Hyperproliferation of hair follicle epithelial cells and basal epidermal keratinocytes-both of ectodermal origin-are the primary characteristics underlying the mutant phenotype. Pathological inflammatory responses have been excluded as a putative cause of the skin and hair disorder. The phenotype of CTSL-deficient mice is reminiscent of the spontaneous mouse mutant furless (fs). Analyses of the ctsl gene of fs mice revealed a G149R mutation inactivating the proteinase activity. CTSL is the first lysosomal proteinase shown to be essential for epidermal homeostasis and regular hair follicle morphogenesis and cycling.

Lymphoid enhancer factor (LEF1), a nuclear mediator of Wnt signaling, is required for the formation of organs that depend on inductive interactions between epithelial and mesenchymal tissues. In previous tissue recombination experiments with normal and Lef1 −/− tooth germs, we found that the effect of LEF1 expression in the epithelium is tissue nonautonomous and transferred to the subjacent mesenchyme. Here we examine the molecular basis for LEF1 function and find that the epithelium of the developmentally arrested Lef1 −/− tooth rudiments fails to express Fgf4, Shh, and Bmp4, but not Wnt10a. We identify the Fgf4 gene as a direct transcriptional target for LEF1 and show that beads soaked with recombinant FGF4 protein can fully overcome the developmental arrest of Lef1 −/− tooth germs. In addition, we find that FGF4 beads induce rapidly the expression of Fgf3 in dental mesenchyme and that both epithelial and mesenchymal FGF proteins induce the delayed expression of Shh in the epithelium. Taken together, these data indicate that a single target of LEF1 can account for the function of LEF1 in tooth development and for a relay of a Wnt signal reception to a cascade of FGF signaling activities, allowing for a sequential and reciprocal communication between epithelium and mesenchyme.

Antigen presentation by major histocompatibility complex (MHC) class II molecules requires the participation of different proteases in the endocytic route to degrade endocytosed antigens as well as the MHC class II-associated invariant chain (Ii). Thus far, only the cysteine protease cathepsin (Cat) S appears essential for complete destruction of Ii. The enzymes involved in degradation of the antigens themselves remain to be identified. Degradation of antigens in vitro and experiments using protease inhibitors have suggested that Cat B and Cat D, two major aspartyl and cysteine proteases, respectively, are involved in antigen degradation. We have analyzed the antigen-presenting properties of cells derived from mice deficient in either Cat B or Cat D. Although the absence of these proteases provoked a modest shift in the efficiency of presentation of some antigenic determinants, the overall capacity of Cat B Major histocompatibility complex (MHC) class II molecules display to CD4 ϩ cells a variety of antigenic peptides generated by degradation of endocytosed proteins (1). MHC class II ␣␤ dimers possess a peptide-binding site designed to accommodate a large variety of peptides differing in sequence and length (2-4). When empty, the peptide-binding site is unstable, rendering ␣␤ dimers prone to aggregation at acidic pH (5) and to dissociation (6). This prevents expression of empty class II molecules at the cell surface, which could interact with polypeptides nonspecifically. Because most antigenic determinants are presented by newly synthesized class II molecules (7, 8), antigen-presenting cells (APC) have devised mechanisms to protect the antigen-binding site of the ␣␤ dimers from both interacting nonspecifically with endoplasmic reticulumresident proteins (9) and from destabilization of the dimer (10). This is accomplished by the association of newly synthesized class II ␣␤ dimers with a third subunit, invariant chain Ii (Ii) (11). A specialized region of Ii, CLIP [from CLass IIassociated Invariant chain Peptide (3)], fills the binding site of ␣␤ dimers as do antigenic peptides (4, 12, 13). The cytoplasmic region of Ii also carries the information necessary to direct the ␣␤Ii complexes to the endocytic compartments where antigenic peptides reside (14, 15). The activation of the peptidebinding properties of class II ␣␤ dimers occurs by degradation of Ii in a stepwise fashion, in which several proteases of the endosomal/lysosomal system are involved (16)(17)(18)(19)(20)(21). This yields ␣␤ molecules associated with CLIP-containing Ii fragments, which are then substituted by antigenic peptides in a reaction facilitated by the MHC class II-like molecule H-2M (in humans, HLA-DM) (22-24).The generation of the antigenic determinants to be presented by MHC class II also requires the action of proteases (8). Endocytosed antigens are degraded in an as yet incompletely characterized series of proteolytic events. It is not clear whether ␣␤ dimers associate with previously formed determinants, or rather bind larger an...

Dilated cardiomyopathy is a frequent cause of heart failure and is associated with high mortality. Progressive remodeling of the myocardium leads to increased dimensions of heart chambers. The role of intracellular proteolysis in the progressive remodeling that underlies dilated cardiomyopathy has not received much attention yet. Here, we report that the lysosomal cysteine peptidase cathepsin L (CTSL) is critical for cardiac morphology and function. Oneyear-old CTSL-deficient mice show significant ventricular and atrial enlargement that is associated with a comparatively small increase in relative heart weight. Interstitial fibrosis and pleomorphic nuclei were found in the myocardium of the knockout mice. By electron microscopy, CTSL-deficient cardiomyocytes contained multiple large and apparently fused lysosomes characterized by storage of electron-dense heterogeneous material. Accordingly, the assessment of left ventricular function by echocardiography revealed severely impaired myocardial contraction in the CTSL-deficient mice. In addition, echocardiographic and electrocardiographic findings to some degree point to left ventricular hypertrophy that most likely represents an adaptive response to cardiac impairment. The histomorphological and functional alterations of CTSL-deficient hearts result in valve insufficiencies. Furthermore, abnormal heart rhythms, like supraventricular tachycardia, ventricular extrasystoles, and first-degree atrioventricular block, were detected in the CTSL-deficient mice.

Summary: Major histocompatibility complex class II antigen presentation requires the participation of lysosomal proteases in two convergent processes. First, the antigens endocytosed by the antigen‐presenting cells must be broken down into antigenic peptides. Second, class II tnolecules are synthesized with their peptide‐binding site blocked by invariant chain (li), and they acquire the capacity to bind antigens only after Ii has been degraded in the compartments where peptides reside. The study of genetically modified tnice deficietit in single lysosomal proteases has allowed us to determine their role in these processes, Cathepsins (Cat) B and D. previously considered major players in MHC class II antigen presentation, are dispensable for degradation of Ii and for generation of several antigenic determinants. By contrast, Cat S plays an essential role in removal of Ii in B cells and dendritic cells, whereas Cat L apparently does so in thymic epithelial cells. Accordingly, the absence of Cat S and L have major consequences for the onset of humoral immtine responses and for T‐cell selection, respectively. It is likely that other as yet uncharacterized lysosomal enzymes also play a role in Ii degradation and in generation of antigenic determinants. Experiments involving drugs that interfere with protein traffic suggest that more than one mechanism for Ii removal, probably involving different proteases, can co‐exist in the same antigen‐presenting cell. These findings may allow the development of protease inhibitors with possible therapeutic applications.