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Identification of a Metalloproteinase-17-mediated cellular signalling circuit in intestinal regeneration and tumorigenesis

Periodic Reporting for period 1 - MetalloGutRepair (Identification of a Metalloproteinase-17-mediated cellular signalling circuit in intestinal regeneration and tumorigenesis)

Reporting period: 2018-04-01 to 2020-03-31

Our digestive tract is continuously working to allow us to incorporate the water and the nutrients that our body needs, keeping pathogens, and possible threats under control. To do so, the intestinal surface is layered by a barrier named the intestinal epithelium. This barrier is bombarded with chemical, mechanical, and biological injuries daily. But, can resist and perform its absorptive role thanks to its high renewal rate. Cells are renewed by younger cells coming from the bottom part of structures called crypts in the epithelium. At the crypts, intestinal stem cells (ISCs), replicate themselves and give rise to daughter cells that will generate all the different epithelial cells (including cells that absorb nutrients, cells that produce mucus and cells that defend the intestine from pathogens). These ISCs need a proper environment to thrive and this is called the niche. The niche is a mix of factors, such as growth factors, fundamental in ISCs maintenance. When this niche is affected, the barrier is compromised and could lead to diseases as intestinal cancer, or impaired renewal after injury. One example of barrier dysfunction occurs in ulcerative colitis or in Chron’s disease (IBD, inflammatory bowel diseases).
Still many aspects of the ISCs niche are unknown. Considering the importance of these factors in human diseases, we highlight the necessity to investigate it in more detail.
Much has been uncovered about the role of epithelial cells in intestinal homeostasis, regeneration, and tumorigenesis, studied almost as a sole entity. In this work, we go a step forward and study other intestinal populations largely understudied, the intestinal muscle. The intestinal muscle is an autonomous musculature (smooth muscle) that is specifically located surrounding the epithelium. The upper part of this muscle is near the bottom of the crypts where the ISCs are located. So, we investigated the regulation of the intestinal epithelium by the muscle, under different circumstances, something that hasn’t been addressed in detail before.
We demonstrated that this muscle has a fundamental role in regulating the ISCs niche. This role is so important that, when we removed a specific protein only present in the muscle, it results in a reduction of the ISCs number and therefore the reparative role of the intestinal barrier was compromised. Because of that, intestinal threats in mice lacking this protein resulted in injuries unable to heal and increased tumour generation.
With these results, our objectives have been achieved. First, we proposed to establish the role of this protein in intestinal inflammation and regeneration. We know today that this protein is necessary during intestinal regeneration after diverse intestinal injuries. Second, we wanted to determine the role of this protein in intestinal tumorigenesis and we demonstrated that its protective against tumour generation, since mice lacking the protein developed a higher number of tumours. Our third objective was to identify the factors that interact with this protein. We have identified a substrate for this protein, that is implicated in intestinal cancers and found several factors that are produced by the muscle and regulate the intestinal epithelium.
In sum, we have demonstrated the importance of the intestinal muscle as an ISCs niche provider, regulating the epithelial function, particularly during intestinal regeneration after injury. This investigation expands the knowledge on the intestinal healing process of crucial importance in human diseases as IBD, intestinal infections, and tumours.
To achieve these objectives, we used in vivo disease models mimicking human pathologies, ex vivo organoids cultures, and in vitro biochemical methods.
First, we cultured intestinal organoids (mini-“gut” structures that grow from intestinal epithelium in a petri dish), and exposed them to intestinal muscle. We showed that the muscle was producing factors that altered the growth and differentiation of the epithelium. We identified these factors by mass spectrometry and performed an RNAseq to identify the specific changes induced by the muscle.
Then we performed an RNAseq comparing muscle and epithelial cells tissue. This experiment showed us that (1) the muscle produces important niche factors (related to BMP, bone morphogenic protein, signaling) and (2) the loss of a muscle protein impacted into epithelial cells affecting BMP signals. By comparing wild-type (WT) mice and mice lacking our protein of interest (knock-out (KO) mice), we determined that KOs had a lower number of ISCs, and this was related to alterations in the BMP signaling pathway.
Then we exposed WT and KO mice to in vivo disease intestinal models. In response to an inflammatory chemical agent called DSS (dextran sodium sulfate) that mimics human IBD, KO mice were unable to heal their epithelium, even in the long term.
When exposed to radiation, KO intestines also showed impaired healing, this time in response to a non-inflammatory insult.
Alterations in the ISCs niche are directly connected to tumour formation. Therefore, we next tested whether the loss of this protein implicated in BMP signaling regulation would be related to intestinal tumours. We used a mouse model, ApcMin, that spontaneously develop intestinal tumours due to impaired ISCs niche signaling. KO mice developed more intestinal tumours. That probed that the loss of a muscular protein also impacted epithelial tumour development.
Finally, we have found a specific substrate for our protein and we are currently analyzing others, to unravel the molecular mechanisms behind the changes that we see in the epithelium.
Since this is basic research, we do not propose any direct exploitation more than increasing the available knowledge on intestinal homeostasis and healing that is so important in human intestinal pathologies.
This work sheds some light on the complex healing processes occurring during intestinal pathologies, including intestinal inflammation, intestinal infections, and cancer; helping scientists to create new ways of treating these important intestinal diseases.
Particularly, we have provided new information beyond the state of the art regarding:
1.- The intestinal muscle is not a mere contractile structure but regulates the epithelium by the production of ISCs niche factors.
2.- Muscle alterations, such as the loss of a specific protein, results in impaired response capacity of the epithelium to injuries that mimic human IBD processes and radiotherapy.
3.- We provide a list of factors produced by the intestinal muscle and we are currently investigating the mechanisms of action.
This work is positively impacting my career development since it has allowed me to learn and mastered in vitro and ex vivo intestinal epithelial cell model systems, and increase my networking and dissemination opportunities by attending several international meetings on the field. Our goal in dissemination is to publish this work in a peer-review international journal to target researchers in the intestinal biology field. The results have been communicated to the scientific community in different conferences, workshops, and meetings and will be published and available to the scientific community in an open-source peer-reviewed journal. We will also outreach the general public by collaborating with NTNU platforms to communicate to different audiences once the work is published.
Small intestinal organoid culture observed at the microscope